Bovine herpesvirus detection and treatment

ABSTRACT

Methods, compositions, devices, and kits are described herein that are useful for detecting BoHV-1 infection in animals and/or for distinguishing animals that may benefit from administration of BoHV-1 tmv vaccine.

This application is U.S. National Stage Application of PCT/2015/043112,filed Jul. 31, 2015, which application claims benefit of the filing dateof U.S. Provisional Patent Application No. 62/032,098, filed Aug. 1,2014, the contents of which applications are specifically incorporatedherein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 23, 2018, isnamed Sequence_Listing_15501094.txt and is 12,288 bytes in size.

BACKGROUND

Bovine herpesvirus type 1 (BoHV-1) is a viral pathogen of cattle thatcan cause severe respiratory tract infections known as infectious bovinerhinotracheitis (IBR), abortion in pregnant cows. BoHV-1 is also asignificant contributor to the development of Bovine respiratory diseasecomplex (BRDC). BRDC is a multifactorial disease in cattle involvinginitial viral respiratory infection that may include BoHV-1, bovinerespiratory syncytial virus (BRSV) and bovine viral diarrheal virus(BVDV) followed by secondary bacterial infection and severebronchopneumonia. BRDC costs the US cattle industry alone more than $1billion annually. The ability of BoHV-1 to immunosuppress infectedcattle, to establish a lifelong latent infection in the trigeminalganglia (TG) of infected animals, to reactivate from latency uponstress, and to be transported anterogradely from neuron cell bodies inthe trigeminal ganglia to axon termini in the nasal and upperrespiratory epithelium followed by replication in the upper respiratorytract predisposes the latently infected animals to BRDC. Therefore,BoHV-1 is considered to be an important initiator of BRDC.

Currently, the only vaccine allowed in EU countries against BoHV-1 isthe entire gE-deleted BoHV-1 vaccine. By using the gE gene-deletedmarker vaccine, BoHV-1 has largely been eradicated in a number ofEuropean countries. The gE-deleted vaccine vaccinated cattle can bedifferentiated from the wild type (wt) BoHV-1-infected cattle by variousassays and, in the field, the gE-deleted BoHV-1 vaccine is being used inconjunction with a serological marker test marketed by IDEXX. Becausethe gE-deleted BoHV-1 vaccine is distinguishable from wild type BoHV-1,it is a “Differentiating Infected from Vaccinated Animals” (DIVA)vaccine. DIVA status for any BoHV-1 vaccine is important for theenforcement of BoHV-1 eradication program, and is necessary formarketing a BoHV-1 vaccine in Europe. As a result, the gE-deleted BoHV-1vaccine is the only BoHV-1 vaccine marketed in Europe.

The inventors have developed a new BoHV-1 vaccine called the BoHV-1 tmvvaccine. Comparative tests indicate that the vaccine efficacy the BoHV-1tmv is significantly better than the gE-deleted BoHV-1 vaccine. Testsalso indicate that the IDEEX test that is currently used to distinguishgE-deleted vaccine vaccinated cattle from wild type (wt) BoHV-1-infectedcattle is not suitable for distinguishing the BoHV-1 tmv-vaccinatedcalves from BoHV-1 wt-infected calves. This may be because BoHV-ltmv hasa carboxy-terminal deletion of amino acids 452-575 but it retainsapproximately two-thirds of the gE coding region. Hence, amino acids1-451 of gE, including the extracellular and transmembrane domains, arepresent in the inventors' BoHV-1 tmv vaccine. Therefore, a serologicalmarker test that would distinguish the BoHV-ltmv vaccinated animals fromthe BoHV-1 wt infected animals is necessary before the improved BoHV-1tmv vaccine can be introduced in the field.

SUMMARY

Described herein is an assay for detection of a highly efficaciousrecombinant BoHV-1 triple mutant virus (BoHV-1 tmv). Compared to wildtype BoHV-1, the BoHV-1 triple mutant virus lacks the immunosuppressivefunctions encoded within UL49.5 (i.e., the BoHV-1 tmv vaccine does nothave UL49.5 amino acids 30-32 and 80-96). In addition, the BoHV-1 tmvvaccine lacks the gE cytoplasmic tail (gE CT residues 451-575), which isassociated with virulence function. In addition, the BoHV-1 tmv vaccinelacks the entire 435 base pair long Us9 open reading frame. Results showthat viruses with deletions of the gE cytoplasmic tail and the Us9 openreading frame do not reactivate from latency in the trigeminal gangliabecause they cannot be transported anterogradely. Therefore, the BoHV-1tmv strain may not shed within bovine nasal secretions following latencyreactivation. Because of these and other properties, the vaccineefficacy the BoHV-1 tmv is significantly better than the BoHV-1gE-deleted virus, and comparative tests demonstrate that this is thecase. For example, the protective efficacy of the BoHV-1 tmv vaccineagainst challenge by wild type BoHV-1 is significantly better thanobserved for the gE-deleted vaccine virus.

One aspect of the invention is a method comprising: (a) contacting atest sample with at least one polypeptide or peptide to form an assaymixture, where the at least one polypeptide or peptide has or includes asequence with at least 95% sequence identity to any of SEQ ID NO:1,4-44, or 45; and (b) detecting or measuring whether a complex betweenthe at least one polypeptide or peptide and antibodies is present in theassay mixture.

Another aspect of the invention is a device comprising a solid surfaceand at least one polypeptide or peptide that has a sequence with atleast 95% sequence identity to any of SEQ ID NO:1, 4-44, or 45. Anotheraspect is a device comprising a solid surface and an antibody thatselectively binds at least one polypeptide or peptide antigen with asequence selected from SEQ ID NO:1, 4-44, or 45.

Another aspect of the invention is an expression cassette or expressionvector that includes a nucleic acid segment encoding a polypeptide orpeptide comprising a sequence with at least 95% sequence identity to anyof SEQ ID NO:1, 4-44, or 45. Another aspect is an expression cassette orexpression vector comprising a nucleic acid segment that includes asequence with at least 95% sequence identity to SEQ ID NO:2 or 3.

Another aspect is a device that includes a solid surface and at leastone polypeptide or peptide comprising or consisting essentially of asequence with at least 95% sequence identity to any of SEQ ID NO:1,4-44, or 45. Another aspect is a kit that includes such a device, andinstructions for using the device.

Another aspect of the invention is a kit that includes instructions foruse of the kit components, and any of the following separately packagedcomponents: (a) at least one polypeptide or peptide comprising orconsisting essentially of a sequence with at least 95% sequence identityto any of SEQ ID NO:1, 4-44, or 45; (b) a binding entity thatspecifically binds to at least one polypeptide or peptide comprising orconsisting essentially of a sequence with at least 95% sequence identityto any of SEQ ID NO:1, 4-44, or 45; (c) a secondary binding entity thatspecifically binds to at least one polypeptide or peptide comprising orconsisting essentially of a sequence with at least 95% sequence identityto any of SEQ ID NO:1, 4-44, or 45; (d) a label or a reagent fordeveloping a signal from a label; or (e) any combination thereof.

DESCRIPTION OF THE FIGURES

FIG. 1A-1F show the design and expression strategy for generating arecombinant gE cytoplasmic tail (CT) polypeptide fragment based upon thepredicted BoHV-1 gE CT amino acid sequence. FIG. 1A is a schematicdiagram the BHV-1 envelope protein, gE, showing the extracellular(Ecto), transmembrane (TM) and cytoplasmic tail (CT) domains. FIG. 1B isthe predicted amino acid sequence of BoHV-1 gE CT domain (BoHV-1 gEresidues 451-570; SEQ ID NO:1). FIG. 1C is the nucleotide sequence ofthe BoHV-1 gE CT coding region, which was codon optimized for E. coliexpression (SEQ ID NO:2). FIG. 1D is the nucleotide sequence of theBoHV-1gE CT coding region fused at the 3′ end with a segment encodingsix histidine residues (SEQ ID NO:3). This construct was placed in thepET-43.1a vector. FIG. 1E is the amino acid sequence (SEQ ID NO:4) ofthe BoHV-1 gE CT-His antigen expressed in E. coli from the pET-43.1a gECT-His expression vector. The estimated isoelectric point (pI) andmolecular weight (MW) of the recombinant BoHV-1 gE CT-His antigen isalso shown. FIG. 1F is a schematic diagram of BoHV-1 U_(L)49.5 Δ30-32CT-null/gE-CTA/Us9Δ virus genome (BoHV-1 tmv) showing (1) a schematic ofthe BoHV-1 genome with the locations of U_(L)49.5 (gN), gE, Us9 andbICP22 open reading frames; (2) a schematic of the BoHV-1 U_(L)49.5430-32 CT-null genome; and (3) a schematic of pBoHV-1 gE-CTA/Us9Δplasmid showing the location and sizes of deleted sequences (gE CT, Us9and Us9-bICP22 intergenic region), the flanking upstream gE (ectodomainand transmembrane) and downstream bICP22 sequences.

FIG. 2A-2D show SDS-PAGE separated immunoblots used to analyze therecombinant BoHV-1 gE-CT His polypeptide (SEQ ID NO:4) and theanti-BoHV-1 gE CT-specific monoclonal antibody MAb 2H8F3 generated asdescribed herein. FIG. 2A shows a Coomassie blue stained 12% SDS-PAGEgel containing the electrophoretically purified gE CT-His (SEQ ID NO:1)polypeptide (lane 1) and the immunoblotting results of the same gECT-His protein-containing lane after reaction with an anti-His antibody(lane 2). FIG. 2B shows a Coomassie blue-stained 12% SDS-PAGE gelcontaining gE CT-His polypeptide electrophoretically separated underreducing (lane 1) and non-reducing (lane 2) conditions. Molecular weightmarkers are shown in lane (M). FIG. 2C shows an immunoblot with thepurified gE CT-His (SEQ ID NO:1) polypeptide in both lanes (1) and (2)after reaction with fluorescently labeled BoHV-1 gE CT-specific MAb2H8F3antibodies. FIG. 2D shows an immunoblot of the purified gE CT-His (SEQID NO:1) polypeptide in both lanes (1) and (2) after reaction withfluorescently labeled anti-GST antibodies.

FIG. 3 shows a MALDI-TOF Mass Spectrometry plot of gE CT-His protein.

FIG. 4A-4B illustrates immunoblotting analysis of the MAb 2HF83 to showits reactivity against BoHV-1 wt and BoHV-1 tmv-infected cell lysates.FIG. 4A shows an immunoblot of mock-, BoHV-1 wt- and BoHV-1 tmv-infectedMDBK cell lysates separated in a 10% SDS-PAGE after reaction with the gECt-specific MAb 2HF83. FIG. 4B shows an immunoblotting analysis of anidentical Western blot with a gE ectodomain-specific rabbit polyclonalantibody. Note that an approximate 68 kD gE CT-specific band is notrecognized by the MAb 2HF83 in FIG. 4A but is recognized by the gEectodomain-specific rabbit polyclonal antibody in FIG. 4B.

FIG. 5 graphically illustrates ELISA epitope mapping results. Theabsorbance at 450 nm observed for reaction of the HRP conjugatedMAb2H8F3 antibody for each peptide library sample is shown. Peptideswith numbers 1-39 (SEQ ID NOs:5-43) are 12-mers with separate sequencesfrom the E. coli expressed recombinant gE CT-His polypeptide. Peptidewell No. 40 contains streptavidin as a blank control. Peptide well No.41 contains purified E. coli expressed recombinant gE CT-His protein,which was used for the production of the MAb2H8F3 antibody. As shown,the MAb2H8F3 antibody was bound to peptide Nos. 16 and 17 (SEQ ID NOs:20 and 21).

FIG. 6 is a schematic diagram of the pET-43.1a(+) expression vector

FIG. 7A-7F are schematic drawings of devices that can be employed fordetecting and monitoring BoHV-1 infection (and for identifying animalsthat would benefit from vaccination with the the BoHV-1 tmv vaccine).FIG. 7A is a schematic drawing of a lateral flow BoHV-1 detectiondevice. FIG. 7B is a schematic drawing of a lateral flow BoHV-1detection device illustrating display of a signal (*****) after testinga bodily fluid sample. FIG. 7C is a schematic drawing of a device withtwo electrical conducting nanowires, for example, an electricaldirect-charge transfer conductometric biosensor. FIG. 7D is a schematicdrawing of the device shown in FIG. 1C illustrating direct chargetransfer (*****) between two electrical conducting nanowires aftertesting a bodily fluid sample. FIG. 7E is a schematic drawing of adevice with a longer and a shorter nanowire. FIG. 7F is a schematicdrawing of a device a longer and a shorter nanowire illustrating that asignal (*****) can be transmitted along a nanowire to a transmitterafter testing a bodily fluid.

FIG. 8 illustrates a device and a method of using a test strip forperforming an assay to detect BoHV-1 infection and/or animals that wouldbenefit from vaccination with the BoHV-1 tmv vaccine.

DETAILED DESCRIPTION

Methods and compositions for detecting and distinguishing BoHV-1infected animals from vaccinated and/or non-infected animals aredescribed herein.

Bovine Herpesvirus Type 1 (BoHV-1)

Bovine herpesvirus type 1 (BHV-1) is a pathogen of cattle that can causesevere respiratory tract infection known as infectious bovinerhinotracheitis (IBR), which can cause abortion in pregnant cows(Kaashoek et al., Vet Rec 139(17):416-21 (1996); Tikoo et al., Adv VirusRes 45:191-223 (1995); Jones & Chowdhury, Animal Health Res Rev8(2):187-205 (2007); U.S. Pat. No. 6,221,360). BoHV-1 is also animportant component of the Bovine Respiratory Disease Complex (BRDC or“Shipping fever”). During primary infection, BoHV-1 replicates in thenasal and upper respiratory tract epithelium and transiently depressescell-mediated immunity and evades the host cytotoxic CD8⁺ T cellrecognition by down regulating the cell surface expression of majorhistocompatibility complex class I (MHC-I) molecules. Thisimmunosuppression combined with lesions in the upper respiratory tractfacilitates secondary bacterial infections and pneumonia. Both IBRdisease and BRDC cause considerable losses for the cattle industryworldwide and cost the US cattle industry at least $1 billion dollarsannually (Tikoo et al., Adv Virus Res 45:191-223 (1995)).

Another problem with BoHV-1 infection in cattle is that the virusestablishes a life-long latency in trigeminal ganglia following theprimary infection (Jones & Chowdhury, Animal Health Res Rev 8(2):187-205(2007)). During the primary infection, the virus enters the sensorynerve endings (axon terminals) of the trigeminal nerve in thenasopharynx and is transported up the axon retrogradely to the neuronalcell bodies in the trigeminal ganglia where BoHV-1 establishes life-longlatency (id.). Periodically throughout the life of the animal, thelatent virus in the trigeminal ganglia reactivates due toimmunosuppression or stress. Following reactivation, the virus istransported down the axon to the primary infection sites in the noseand/or eye. The viral infection causes ocular and nasal virus shedding(id.), which facilitates virus transmission to other cattle. Thus thisreactivation followed by shedding maintains an ongoing viral infectionin susceptible cattle populations.

In BoHV-1-infected cells, envelope protein U_(L)49.5 (a BoHV-1 homologof envelope glycoprotein N (gN)) was found to block the Transporterassociated with antigen presentation (TAP) function required for thedisplay of viral peptides on the cell surface. To promote the immuneresponse from the host, the MHC-I molecules must be loaded with viralpeptides. However, BoHV-1 U_(L)49.5 binds to the TAP complex, blocks TAPconformational changes, and degrades the TAP (Lipinska et al., J Virol80(12):5822-32 (2006)). Consequently, peptides are not loaded onto theMHC-I molecules in the endoplasmic reticulum, which is required for theMHC-I transport to cell surface and cell surface expression. Thisresults in transient MHC-I down-regulation in a susceptible host andhelps the virus evade destruction by the CD8+ T cells at an early stageof virus infection of the host.

BoHV-1 mutants have been made and analyzed for use as vaccines. Onecommercially available vaccine is based on a deletion of the completeglycoprotein E (gE) gene. Following intranasal infection, both thegE-deleted (the entire gE gene deleted) and the inventors' gEcytoplasmic tail (CT)-truncated BHV-1 recombinant viruses weredetermined to be equally attenuated in calves and to have defectiveanterograde axonal transport (Liu et al., J Virol 82(15):7432-42 (2008);Chowdhury et al., J. Neurovirol. 17:457-465 (2010)). Therefore,following dexamethasone-induced reactivation in the trigeminal ganglia,no nasal virus shedding in calves infected with either recombinant viruswas seen (Liu et al., J Virol 82(15):7432-42 (2008)). Importantly, BHV-1gE cytoplasmic tail-deleted virus-infected calves have two-fold higherserum neutralizing titers relative to calves infected with the entiregE-deleted virus (id.).

BoHV-1 gE

The BoHH-1 genome consists of a linear double-stranded DNA molecule ofabout 135.3 kb that encodes for approximately 70 proteins. Twelve ofthese proteins (gB, gC, gD, gE, gG, gI, gH, gK, gL, gM, UL49.5 and Us9)are envelope proteins, and the gB, gC, gD, gE, gG, gI, gH, gK, gL, gM,protein are glycosylated envelope proteins. Envelope glycoproteins gEand gI form complexes and gE is involved in viral intercellular spread(cell-to-cell spread). The BoHV-1 gE open reading frame (ORF) ispredicted to contain 575 amino acid (aa) residues with a 28 amino acidcleavable signal sequence. The structure of glycoprotein E (gE)corresponds to a type I transmembrane glycoprotein. The gE glycoproteincontains three distinct domains: a 387 amino acid long hydrophilicextracellular domain (ecto-domain), a 33 amino acid long hydrophobictransmembrane domain, and a 125 amino acid long highly chargedcytoplasmic domain/tegument domain. The mature gE is phosphorylated andglycosylated with an apparent molecular mass of about 92 kD.

The BoHV-1 tmv vaccine previously generated by the inventors is arecombinant BoHV-1 triple mutant virus that lacks the immunosuppressive(deletion of UL49.5 residues 30-32 and 80-96), the virulence (deletionof the gE cytoplasmic tail residues 452-575), and the anterogradeneuronal transport (deletion of gE cytoplasmic tail and the entire 435bp long Us9 ORF) properties (Chowdhury et al., Vaccine 32(39):4909-4915(2014)) (see, FIG. 1F). Although the inventors' BoHV-1 tmv vaccine ismissing a C-terminal part of the glycoprotein E cytoplasmic tail, itretains and expresses the N-terminal 1-451 amino acids of gE, includingthe extracellular and transmembrane domains of the BoHV-1 glycoproteinE.

The compositions, methods, kits and devices described herein are usefulfor distinguishing animals that are vaccinated with the BoHV-1 tmvvaccine from those that are infected with BoHV-1. In addition, thecompositions, methods, kits and devices described herein are also usefulfor distinguishing animals that not infected with BoHV-1 from those thatare infected. Such compositions, methods, kits and devices can detectantibodies and/or antigens that are present in BoHV-1-infected animalsbut they do not detect any such antibodies or antigens in non-infectedanimals or in animals that are vaccinated with the BoHV-1 tmv vaccine.

In particular, the compositions, methods, kits and devices describedherein detect a portion of glycoprotein E that is not part of the BoHV-1tmv vaccine—the glycoprotein E cytoplasmic tail. The structure ofglycoprotein E is schematically shown in FIG. 1A. The part ofglycoprotein E that is missing in the BoHV-1 tmv vaccine is the 451-575amino acid glycoprotein E cytoplasmic tail (gE CT), with the sequenceshown below (SEQ ID NO:1; FIG. 1B).

  1 ASQKRTYDIL NPFGPVYTSL PTNEPLDVVV PVSDDEFSLD  41EDSFADDDSD DDGPASNPPA DAYDLAGAPE PTSGFARAPA  81NGTRSSRSGF KVWFRDPLED DAAPARTPAA PDYTVVAARL 121 KSILR

This missing cytoplasmic tail domain of the BoHV-1 gE protein is usefulas an antigen for developing assays that detect BoHV-1 infection,thereby identifying infected animals. Non-infected animals and animalsthat have been vaccinated with the BoHV-1 tmv vaccine do not developantibodies against the cytoplasmic tail domain of the BoHV-1 gE proteinand are not detected with the gE CT antigen-based assays describedherein.

A nucleic acid that encodes the SEQ ID NO:1 glycoprotein E cytoplasmictail is shown below (SEQ ID NO:2; FIG. 1C).

  1 GCATCGCAAA AGCGTACCTA TGATATTCTG AACCCGTTTG  41GTCCGGTCTA CACGAGCCTG CCGACGAACG AACCGCTGGA  61TGTTGTTGTG CCTGTTAGTG ATGACGAATT TTCCCTGGAT 121GAAGACTCAT TCGCCGATGA CGATTCGGAC GATGACGGTC 161CGGCAAGCAA CCCGCCGGCA GATGCTTATG ATCTGGCAGG 201TGCACCGGAA CCGACCTCTG GTTTTGCACG TGCTCCGGCG 241AATGGCACGC GTAGCTCTCG CTCCGGTTTT AAAGTCTGGT 281TCCGCGATCC GCTGGAAGAT GACGCGGCCC CGGCGCGTAC 321CCCGGCGGCA CCGGACTACA CCGTGGTTGC GGCCCGTCTG 361 AAGAGCATCC TGCGT

A variant BoHV-1 cytoplasmic tail domain with an N-terminal methionineand an N-terminal six histidine tag is also useful an antigen and isreadily detectable/isolatable due to the presence of the histidine tag.The sequence of this polypeptide is shown below (SEQ ID NO:4; FIG. 1E).

  1 MHHHHHHASQ KRTYDILNPF GPVYTSLPTN EPLDVVVPVS  41DDEFSLDEDS FADDDSDDDG PASNPPADAY DLAGAPEPTS  81GFARAPANGT RSSRSGFKVW FRDPLEDDAA PARTPAAPDY 121 TVVAARLKSI LR

An E. coli codon-optimized nucleic acid sequence that encodes the SEQ IDNO:4 polypeptide is shown below (SEQ ID NO:3; FIG. 1D).

  1 CATATGCATC ACCACCATCA CCACGCATCG CAAAAGCGTA  41CCTATGATAT TCTGAACCCG TTTGGTCCGG TCTACACGAG  81CCTGCCGACG AACGAACCGC TGGATGTTGT TGTGCCTGTT 121AGTGATGACG AATTTTCCCT GGATGAAGAC TCATTCGCCG 161ATGACGATTC GGACGATGAC GGTCCGGCAA GCAACCCGCC 201GGCAGATGCT TATGATCTGG CAGGTGCACC GGAACCGACC 241TCTGGTTTTG CACGTGCTCC GGCGAATGGC ACGCGTAGCT 281CTCGCTCCGG TTTTAAAGTC TGGTTCCGCG ATCCGCTGGA 321AGATGACGCG GCCCCGGCGC GTACCCCGGC GGCACCGGAC 361TACACCGTGG TTGCGGCCCG TCTGAAGAGC ATCCTGCGTT 401 AATGACTCGA G

The sequences of the polypeptides, peptides, and nucleic acids describedherein can vary somewhat from the sequences recited herein for the wildtype BoHV-1 virion. In addition, the sequences of the polypeptides,peptides, and nucleic acids employed in the methods, compositions,devices, and kits described herein can also vary. For example, thenucleic acids encoding the polypeptides and peptides described hereincan be codon-optimized for expression in a variety of host cells (e.g.,in various prokaryotic or eukaryotic host cell species or strains).Hence, the polypeptides, peptides, and nucleic acids employed and/ordetected herein can have at least 75% sequence identity, or at least 80%sequence identity, or at least 85% sequence identity, or at least 90%,or at least 95% sequence identity to any of SEQ ID NO:1-45.

Sequence identity and sequence variation can be evaluated using sequenceanalysis software (e.g., via the NCBI tools, or the Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue. Madison, Wis. 53705).Software can be employed to match similar sequences by assigning degreesof sequence identity to various substitutions, deletions, insertions,and other modifications. Conservative amino acid substitutions, forexample, typically include substitutions within the following groups:the group of glycine, alanine; valine, isoleucine, and leucine; thegroup of aspartic acid, glutamic acid, asparagine, and glutamine; thegroup of serine and threonine; the group of lysine and arginine; and thegroup of phenylalanine and tyrosine.

Expression Cassettes or Vectors

The nucleic acids with sequences that are at least 95% identical to SEQID NO:2 or 3 are useful for efficiently expressing the glycoprotein Ecytoplasmic tail, which can be used as antigen (or to generateantibodies) for detecting BoHV-1 infection and distinguishing animalsthat have been vaccinated with the BoHV-1 tmv vaccine from those thatneed to be vaccinated.

Antigenic peptide fragments of the glycoprotein E cytoplasmic tail arealso useful as antigens for detection of BoHV-1 infection, and forgenerating antibodies for detection of BoHV-1 and BoHV-1 infection.Examples of such antigenic fragments of the BoHV-1 glycoprotein Ecytoplasmic tail include polypeptides and/or peptides with a sequencethat is at least 95% identical to any of SEQ ID NOs: 1, 4, 5-44 or 45.These polypeptides and/or peptides can be encoded within expressioncassettes or expression vectors that include one or more nucleic acidsegments.

For example, one of skill in the art can prepare an expression cassetteor expression vector that can express one or more encoded BoHV-1glycoprotein E cytoplasmic tail polypeptides or peptides. Host cells canbe transformed by the expression cassette or expression vector, and theexpressed polypeptides or peptides can be isolated therefrom. Someprocedures for making such genetically modified host cells are describedbelow.

Promoters: Nucleic acids or nucleic acid segments encoding the BoHV-1glycoprotein E cytoplasmic tail can be operably linked to a promoter,which provides for expression of an mRNA encoding the BoHV-1glycoprotein E cytoplasmic tail polypeptides or peptides. The promotercan be a promoter functional in a host cell such as a viral promoter, abacterial promoter or a mammalian promoter. The promoter can be aheterologous promoter. As used herein, “heterologous” when used inreference to a gene or nucleic acid refers to a gene or nucleic acidthat has been manipulated in some way. For example, a heterologouspromoter is a promoter that contains sequences that are not naturallylinked to an associated coding region. Thus, a heterologous promoter isnot the same as the natural BoHV-1 promoter that drives expression ofglycoprotein E.

A BoHV-1 glycoprotein E cytoplasmic tail nucleic acids are operablylinked to the promoter when so that the BoHV-1 glycoprotein Ecytoplasmic tail coding region is located downstream from the promoter.The operable combination of the promoter with the BoHV-1 glycoprotein Ecytoplasmic tail coding region is a key part of the expression cassetteor expression vector.

Promoter regions are typically found in the flanking DNA upstream fromthe coding sequence in both prokaryotic and eukaryotic cells. A promotersequence provides for regulation of transcription of the downstream genesequence and typically includes from about 50 to about 2,000 nucleotidebase pairs. Promoter sequences also contain regulatory sequences such asenhancer sequences that can influence the level of gene expression. Someisolated promoter sequences can provide for gene expression ofheterologous DNAs, that is a DNA different from the native or homologousDNA.

Promoter sequences are also known to be strong or weak, or inducible. Astrong promoter provides for a high level of gene expression, whereas aweak promoter provides a very low level of gene expression. An induciblepromoter is a promoter that provides for the turning on and off of geneexpression in response to an exogenously added agent, or to anenvironmental or developmental stimulus. For example, a bacterialpromoter such as the P_(tac) promoter can be induced to vary levels ofgene expression depending on the level of isothiopropylgalactoside addedto the transformed cells. Promoters can also provide for tissue specificor developmental regulation. An isolated promoter sequence that is astrong promoter for heterologous DNAs is advantageous because itprovides for a sufficient level of gene expression for easy detectionand selection of transformed cells and provides for a high level of geneexpression when desired. In some embodiments, the promoter is aninducible promoter and/or a tissue-specific promoter.

Examples of promoters that can be used include, but are not limited to,the T7 promoter (e.g., optionally with the lac operator), the CaMV 35Spromoter (Odell et al., Nature. 313:810-812 (1985)), the CaMV 19Spromoter (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nospromoter (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749(1987)), Adh1 promoter (Walker et al., Proc. Natl. Acad. Sci. USA.84:6624-6628 (1987)), sucrose synthase promoter (Yang et al., Proc.Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin promoter,ubiquitin promoter, actin promoter (Wang et al., Mol. Cell. Biol.12:3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)),PEPCase promoter (Hudspeth et al., Plant Molecular Biology. 12:579-589(1989)), the CCR promoter (cinnamoyl CoA:NADP oxidoreductase, EC1.2.1.44) isolated from Lollium perenne, (or a perennial ryegrass)and/or those associated with the R gene complex (Chandler et al., ThePlant Cell. 1:1175-1183 (1989)).

Other constitutive or inducible promoters can be used with or withoutassociated enhancer elements. Examples include a baculovirus derivedpromoter, the p10 promoter. Plant or yeast promoters can also be used.

Alternatively, novel tissue specific promoter sequences may be employedin the practice of the present invention. Coding regions from aparticular cell type or tissue can be identified and the expressioncontrol elements of those coding regions can be identified usingtechniques available to those of skill in the art.

The nucleic acid encoding the BoHV-1 glycoprotein E cytoplasmic tail orpeptide therefrom can be combined with the promoter by available methodsto yield an expression cassette, for example, as described in Sambrooket al. (Molecular Cloning: A Laboratory Manual. Second Edition (ColdSpring Harbor, N.Y.: Cold Spring Harbor Press (1989); Molecular Cloning:A Laboratory Manual. Third Edition (Cold Spring Harbor, N.Y.: ColdSpring Harbor Press (2000)). For example, a plasmid containing apromoter such as the T7-lac promoter can be constructed or obtained fromSnap Gene (see, e.g., website at snapgene.com/resources/plasmidfiles/pet_and_duet_vectors_%28novagen%29/pET-43.1a%28+%29/). These andother plasmids are constructed to have multiple cloning sites havingspecificity for different restriction enzymes downstream from thepromoter. The nucleic acid encoding the BoHV-1 glycoprotein Ecytoplasmic tail or peptide therefrom can be subcloned downstream fromthe promoter using restriction enzymes and positioned to ensure that theDNA is inserted in proper orientation with respect to the promoter sothat the DNA can be expressed as sense RNA.

Expression cassettes that include a promoter operably linked to a BoHV-1glycoprotein E cytoplasmic tail polypeptide or peptide coding region caninclude other elements such as a segment encoding 3′ nontranslatedregulatory sequences, and restriction sites for insertion, removal andmanipulation of segments of the expression cassettes. The 3′nontranslated regulatory DNA sequences can act as a signal to terminatetranscription and allow for the polyadenylation of the resultant mRNA.The 3′ nontranslated regulatory DNA sequence preferably includes fromabout 300 to 1,000 nucleotide base pairs and contains prokaryotic oreukaryotic transcriptional and translational termination sequences.Various 3′ elements that are available to those of skill in the art canbe employed. These 3′ nontranslated regulatory sequences can be obtainedas described in An (Methods in Enzymology. 153:292 (1987)). Many such 3′nontranslated regulatory sequences are already present in plasmidsavailable from commercial sources such as Clontech, Palo Alto, Calif.The 3′ nontranslated regulatory sequences can be operably linked to the3′ terminus of a BoHV-1 glycoprotein E cytoplasmic tail polypeptide orpeptide coding region by available methods.

Once the nucleic acid encoding the BoHV-1 glycoprotein E cytoplasmictail or peptide therefrom is operably linked to a promoter (e.g., andother selected elements), the expression cassette so formed can besubcloned into a plasmid or other vector (e.g., an expression vector).Such expression vectors can have a prokaryotic or eukaryotic replicationorigin, for example, to facilitate episomal replication in bacterial,vertebrate and/or yeast cells.

Examples of vectors that provide for easy selection, amplification, andtransformation of the expression cassette in prokaryotic and eukaryoticcells include pET-43.1a(+), pUC-derived vectors such as pUC8, pUC9,pUC18, pUC19, pUC23, pUC119, and pUC120, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. Theadditional DNA sequences include origins of replication to provide forautonomous replication of the vector, additional selectable markergenes, such as antibiotic or herbicide resistance, unique multiplecloning sites providing for multiple sites to insert DNA sequences,and/or sequences that enhance transformation of prokaryotic andeukaryotic cells.

In order to improve identification of transformed cells, a selectable orscreenable marker gene can be employed in the expression cassette orexpression vector. “Marker genes” are genes that impart a distinctphenotype to cells expressing the marker gene and thus allow suchtransformed cells to be distinguished from cells that do not have themarker. Such genes may encode either a selectable or screenable marker,depending on whether the marker confers a trait which one can ‘select’for by chemical means, i.e., through the use of a selective agent (e.g.,an antibiotic), or whether it is simply a trait that one can identifythrough observation or testing, i.e., by ‘screening’ (e.g., the R-locustrait). Of course, many examples of suitable marker genes are known tothe art and can be employed in the practice of the invention.

Included within the terms selectable or screenable “marker” genes aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or secretable enzymes that can be detected bytheir catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; and proteins that are inserted or trapped in the cell wall (e.g.,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, an ampicillin gene, whichcodes for the ampicillin antibiotic. Other examples include a neo gene(Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985)) which codes forkanamycin resistance and can be selected for using kanamycin, G418, andthe like; a mutant acetolactate synthase gene (ALS) which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (European Patent Application 154,204 (1985)); amethotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem.263:12500-12508 (1988)); a dalapon dehalogenase gene that confersresistance to the herbicide dalapon; a mutated anthranilate synthasegene that confers resistance to 5-methyl tryptophan; a β-galactosidasegene, which encodes an enzyme for which there are chromogenicsubstrates; a luciferase (lux) gene (Ow et al., Science.234:856-859.1986), which allows for bioluminescence detection; or anaequorin gene (Prasher et al., Biochem. Biophys. Res. Comm.126:1259-1268 (1985)), which may be employed in calcium-sensitivebioluminescence detection, or a green or yellow fluorescent protein gene(Niedz et al., Plant Cell Reports. 14:403 (1995).

The expression cassettes and/or expression vectors can be introducedinto a recipient host cell to create a transformed cell by availablemethods. The frequency of occurrence of cells taking up exogenous(foreign) DNA can be low, and it is likely that not all recipient cellsreceiving DNA segments or sequences will result in a transformed cellwherein the DNA is stably integrated into the host cell chromosomeand/or expressed. Some may show only initial and transient geneexpression. However, cells from virtually any species can be stablytransformed, and those cells can be utilized to generate antigenicpolypeptides or peptides.

Transformation of the host cells with expression cassettes or expressionvectors can be conducted by any one of a number of methods available tothose of skill in the art. Examples are: transformation by direct DNAtransfer into host cells by electroporation, direct DNA transfer intohost cells by PEG precipitation, direct DNA transfer to plant cells bymicroprojectile bombardment, and calcium chloride/heat shock.

Methods such as microprojectile bombardment or electroporation can becarried out with “naked” DNA where the expression cassette may be simplycarried on any E. coli-derived plasmid cloning vector. In the case ofviral vectors, it is desirable that the system retain replicationfunctions, but lack functions for disease induction.

Once the glycoprotein E cytoplasmic tail polypeptide or peptideexpression cassette or vector has been constructed and introduced into ahost cell, the host cells can be screened for the ability to express theencoded glycoprotein E cytoplasmic tail polypeptide or peptide byavailable methods. For example, when the glycoprotein E cytoplasmic tailpolypeptide or peptide has a poly-histidine tag, and the His-taggedglycoprotein E cytoplasmic tail polypeptide or peptide can be detectedor isolated by use of anti-His tag antibodies. In another example,glycoprotein E cytoplasmic tail polypeptides or peptides can be detectedusing antibodies that bind to the polypeptides or peptides (e.g., viawestern blot or ELISA). Nucleic acids encoding the glycoprotein Ecytoplasmic tail polypeptide or peptide can be detected by Southernblot, or nucleic acid amplification using complementary probes and/orprimers.

Immunological Assays

The immunological assays described herein can detect BoHV-1 infection bydetecting BoHV-1 antigens or antibodies in test samples obtained fromanimals.

No particular limitation is imposed on the type of the immunologicalassay method of the present invention, so long as the method involvesformation of an antigen-binding entity (e.g., antibody) reaction.However, the assay method preferably includes a step for the formationof a complex between a BoHV-1 cytoplasmic tail antigen (or a peptideantigen thereof), and at least one antibody or binding entity.

Antibodies and antigens in test samples can be detected by techniquesavailable in the art, such as radioimmunoassay, enzyme-linkedimmunosorbent assay (ELISA), “sandwich” immunoassay, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotopelabels), western blot, agglutination assay (e.g., gel agglutinationassay, hemagglutination assay and etc.), complement fixation assay,immunofluorescence assay, sandwich ELISA, immunoturbidimetry (TIA orLTIA (latex turbidimetric immunoassay)), immunochromatography, andimmunoelectrophoresis assay and the like.

Test samples for detecting BoHV-1 antigens or antibodies can includetissues or bodily fluids collected from an animal such as whole blood,blood fractions, serum, nasal secretions, mucus, saliva, urine, feces,lung fluids, lung tissues, and the like. The test sample can bepartially purified, filtered, fractionated (e.g., a supernatant orprecipitate), or unpurified. For example, one convenient test sample iswhole blood or serum.

In some cases, assays can be used to detect antibodies against theBoHV-1 cytoplasmic tail (or a peptide thereof), where the antibodies arepresent in serum samples obtained from BoHV-1 infected animals.

For example, antibody binding can be detected by use of ELISA whereantibodies that can be present in a test sample bind a glycoprotein Ecytoplasmic tail polypeptide or peptide (e.g., any polypeptide orpeptide with SEQ ID NOs: 1, 4-44, or 45). Such antibodies can detectedin bodily fluids, including but are not limited to serum or plasmasamples. A device can be used for detecting by ELISA, for example, wherean antigen is immobilized on a solid surface such as a test strip,lateral flow device, chip, or microtiter plate. The antigen can be aglycoprotein E cytoplasmic tail polypeptide or peptide (e.g., anypolypeptide or peptide with SEQ ID NOs: 1, 4-44, 45, or a combinationthereof). When antibodies are present in the test sample, thoseantibodies react with the immobilized antigen, and block binding by alabeled marker indicator binding entity. The marker indicator bindingentity is specific for the immobilized antigen (i.e., the BoHV-1glycoprotein E cytoplasmic tail polypeptide or peptide thereof) andbinds to the immobilized antigen.

As described in the Examples, a competitive ELISA was developed wheretest sera were incubated in glycoprotein E cytoplasmic tail-His (gECT-His) antigen-coated wells. After washing off the unbound sera, theindicator marker antibody was added, the plates are incubated again toallow any binding that may occur between the gE CT-His antigen coatingthe wells and the indicator marker antibody. The wells are then washedand the signal from an HRP-indicator marker antibody is observed. Testsera containing antibodies against BoHV-1 (i.e., sera from BoHV-1infected animals) have a lower HRP signal than sera without suchantibodies (i.e., uninfected animals), because the antibodies that theinfected animals produce are unlabeled and will block the later bindingby the indicator marker antibody to the gE CT-His antigen.

Hence, one method of detecting BoHV-1 infection can include (a)incubating a test sample in a device that includes aBoHV-1 (e.g., gE CT)antigen immobilized to a solid surface; (b) removing unbound testsample; (c) contacting the antigen immobilized on the solid surface witha labeled marker indicator binding entity; (d) removing unbound labeledmarker indicator binding entity; and (e) quantifying the amount oflabeled marker indicator binding entity bound to the immobilizedantigen. High levels of antibodies in a test sample leads to a lowersignal from the labeled marker indicator binding entity because theantibodies in the test sample block binding to the antigen by thelabeled marker indicator binding entities. Hence, a test sample thatexhibits a strong signal indicates that the animal from which the samplewas obtained is not infected with BoHV-1. Such animals can be vaccinatedwith the BoHV-1 tmv vaccine, if they have not already been sovaccinated.

Another method of detecting BoHV-1 infection can include (a) incubatinga test sample in a device that includes aBoHV-1 (e.g., gE CT) antigen;and (b) observing whether a signal is detectable, where the signalindicates that a complex has formed between the antigen and antibodiesin the test sample. The method can optionally include quantifying theamount of signal.

The amount of labeled marker indicator binding entity bound to theantigen can be quantified by obtaining a quantified signal from thelabel bound to the marker indicator binding entity bound. For example,when the marker indicator binding entity is attached to a colored labelor to a label that can give rise to a visually detectable signal, thesignal can be quantified by measuring the amount of light transmitted oremitted (e.g., fluorescence or luminescence), or the amount of lightabsorbed. The Examples provided describe use of a horse radishperoxidase (HRP) labeled antibody, which is useful as a labeled markerindicator binding entity for this type of assay. When such anHRP-labeled marker indicator binding entity is employed, the amount oflabeled marker indicator binding entity bound to the immobilized antigencan be determined by incubating the device in an HRP substrate fordetecting the absorbance of the colored product that forms by action ofthe HRP enzyme. Such HRP substrates can include 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (absorption at410 nm and 650 nm), o-phenylenediamine dihydrochloride (absorbancemaximum of 492 nm), or 3,3′,5,5′-tetramethylbenzidine (absorption maximaat 370 nm and 652 nm, which changes to an absorption maxima of 450 nmwhen sulfuric or phosphoric acid is added to stop the HRP reaction).

For example, when the optical density of test samples are measured andcompared to a negative controls the following algorithms can be used tocalculate either a sample/negative control (S/N) ratio or a percentinhibition using the following algorithms:

${{Percent}\mspace{14mu}{inhibition}} = {\left( \frac{{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} - {{OD}\mspace{14mu}{sample}}}{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right) \times 100}$${S\text{/}N\mspace{14mu}{ratio}} = \left( \frac{{OD}\mspace{14mu}{sample}}{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right)$

If the S/N ratio was less than or equal to 0.6, the sample wasclassified as positive for the presence of anti-BoHV-1 antibodies (i.e.,the animal from which the serum was obtained was infected with BoHV-1).If the S/N ratio was greater than 0.7, the sample was classified asnegative for anti-BoHV-1 antibodies (i.e., the animal from which theserum was obtained was not infected with BoHV-1). If the SN ratio wasbetween 0.6-0.7, then the sample was classified as suspect. The test canbe repeated using new samples when a suspect sample is detected, or theanimal can be vaccinated or treated for BoHV-1 infection.

In another example, a sandwich ELISA method can be employed, where aprimary binding entity (e.g., anti-gE CT monoclonal antibody) isimmobilized onto solid substrate (e.g., a 96-well plate), and abiological sample (blood, serum, plasma, pharyngeal swab, urine) can beadded to the substrate for contact and binding with the immobilizedbinding entity. After incubation, the plate is washed with anappropriate buffer, and then a labeled secondary binding entity (e.g., alabeled anti-gE CT binding entity) is reacted with the complex. When,for example, the label is HRP, biotin, peroxidase-labeled avidin (orstreptavidin) and an appropriate reaction substrate (e.g., TBM) is addedfor color development. After a certain period of time, colorimetricdetermination is carried out at a specific wavelength (e.g., 450 nm).

Antibodies and/or Binding Entities

As used herein, “binding entities” include any molecule that canspecifically bind to a gE cytoplasmic domain or peptide thereof. Eachbinding entity binds its target gE cytoplasmic domain or peptide thereofwith specificity. Binding entities are typically binding regions ofaffinity molecules available in the biological sciences including, butnot limited to, antibodies, antibody fragments, leucine zippers,histones, complementary determining regions (CDRs), single chainvariable fragments (scFvs), receptors, ligands, aptamers, lectins,nucleic acid probes and the like. Binding entities can include bindingregions that are generated, for example, from full sized versions of anaffinity molecule, fragments of an affinity molecule, or the smallestportion of the affinity molecule providing binding that is useful in thedetection of a target of interest (a gE cytoplasmic domain or peptidethereof).

The methods, compositions, devices and kits described herein can includebinding entities which are members of the immunoglobulin family ofproteins, or derivatives thereof. For example, the binding entity can bea complete immunoglobulin or antibody, a fragment, a single chainvariable fragment (scFv), a heavy or light chain variable region, a CDRpeptide sequence, and/or the like.

As used herein, “antibody” refers to an immunoglobulin molecule, andfragments thereof, which are immunologically reactive with a particularantigen. The term “antibodies” refers to a plurality of such moleculesand is not limited to homogeneous populations of a single type ofantibody. The term “antibody” also includes genetically engineered formssuch as chimeric antibodies, heteroconjugate antibodies (e.g.,bispecific antibodies), and recombinant single chain Fv fragments(scFv), and disulfide stabilized (dsFv) Fv fragments (see, for exampleU.S. Pat. No. 5,747,654). The term “antibody” also includes antigenbinding forms of antibodies (e.g., Fab′, F(ab′)2, Fab, Fv and IgG. Seealso, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.). The term “antibody,” includes immunologically-activefragment of an immunoglobulin molecule such as the Fab or F(ab′)2fragment generated by, for example, cleavage of the antibody with anenzyme such as pepsin or co-expression of an antibody light chain and anantibody heavy chain in bacteria, yeast, insect cell or mammalian cell.The antibody can also be an IgG, IgD, IgA, IgE or IgM antibody.

Antibodies for use in the methods, compositions, kits and devicesdescribed herein can be obtained commercially or can be generated byavailable methods. Methods of making binding entities, antibodies, andantibody fragments are available in the art (see for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, (1988), specifically incorporated herein by reference in itsentirety). For example, antibodies suitable for use the devices can beobtained by immunizing an animal such as a rabbit, goat, sheep, horse,or guinea pig. Such antibodies are present in the blood (e.g., serum) ofimmunized animals.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression of nucleic acids encoding the antibodyfragment in a suitable host. Antibody fragments can be obtained bypepsin or papain digestion of whole antibodies conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment described as F(ab′)2.This fragment can be further cleaved using a thiol reducing agent, andoptionally using a blocking group for the sulfhydryl groups resultingfrom cleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. Nos. 4,036,945 and 4,331,647, andreferences contained therein. These patents are hereby incorporatedherein by reference in their entireties.

A number of proteins can serve as protein scaffolds to which bindingdomains can be attached and thereby form a suitable binding entity. Thebinding domains bind or interact with gE cytoplasmic domains while theprotein scaffold merely holds and stabilizes the binding domains so thatthey can bind. A number of protein scaffolds can be used. For example,phage capsid proteins can be used. See Review in Clackson & Wells,Trends Biotechnol. 12:173-184 (1994). Phage capsid proteins have beenused as scaffolds for displaying random peptide sequences, includingbovine pancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433(1992)), human growth hormone (Lowman et al., Biochemistry30:10832-10838 (1991)), Venturini et al., Protein Peptide Letters1:70-75 (1994)), and the IgG binding domain of Streptococcus (O'Neil etal., Techniques in Protein Chemistry V (Crabb, L., ed.) pp. 517-524,Academic Press, San Diego (1994)). These scaffolds have displayed asingle randomized loop or region that can be modified to include bindingdomains for the gE cytoplasmic domain or a peptide thereof.

Fibronectin type III domain has also been used as a protein scaffold toserve as a binding entity platform. Fibronectin type III is part of alarge subfamily (Fn3 family or s-type Ig family) of the immunoglobulinsuperfamily. Sequences, vectors and cloning procedures for using such afibronectin type III domain as a protein scaffold portion of a bindingentity (e.g. that includes CDR peptides) are provided, for example, inU.S. Patent Application Publication 20020019517. See also, Bork, P. &Doolittle, R. F. (1992) Proposed acquisition of an animal protein domainby bacteria. Proc. Natl. Acad. Sci. USA 89, 8990-8994; Jones, E. Y. Theimmunoglobulin superfamily Curr. Opinion Struct. Biol. 3, 846-852(1993); Bork, P., Hom, L. & Sander, C. (1994) The immunoglobulin fold.Structural classification, sequence patterns and common core. J. Mol.Biol. 242, 309-320; Campbell, I. D. & Spitzfaden, C. (1994) Buildingproteins with fibronectin type III modules Structure 2, 233-337; Harpez,Y. & Chothia, C. (1994).

It can be useful to employ a binding entity that binds to a gEcytoplasmic domain or peptide thereof with specificity. For example, thebinding entity can have an affinity for an gE cytoplasmic domain orpeptide thereof where the affinity is about 1×10⁷ M⁻¹ to about 1×10¹⁰M⁻¹ or about 1×10⁸ M⁻¹ to about 1×10⁹ M⁻¹. For example, the affinity canbe at least about 1×10⁷ M⁻¹, at least about 1×10⁸ M⁻¹, at least about1×10⁹ M⁻¹, or at least about 1×10¹⁰ M⁻¹. The affinity of a bindingentity can be measured by detecting and quantifying the formation of abinding entity-gE cytoplasmic domain complex (or a binding entity-gEcytoplasmic domain peptide complex), generally referred to as anantigen-antibody complex [Ag−Ab]. The formation of such anantigen-antibody complex [Ag−Ab] is illustrated by the followingreaction.Ab+Ag⇄AbAgThe formation of such an Ag−Ab complex is therefore at equilibrium withits dissociation, and the equilibrium association constant (KA) of thecomplex can be calculated as follows:KA=1/kd=[Ag−Ab]/[Ag][Ab]As used herein, the term “binds specifically” or “specifically binds,”in reference to a binding entity or antibody interaction with an gEcytoplasmic domain or peptide thereof, means that the binding entity orantibody binds with a particular antigen (e.g., gE cytoplasmic domain orpeptide thereof with any of SEQ ID NO:5-45) without substantiallybinding to other BoHV-1 proteins or peptides thereof (including thenon-cytoplasmic tail portion of the gE protein).

For example, in some embodiments, binding entities can be employed inthe methods, compositions, kits and devices described herein that bindwith greater affinity or selectivity to gE cytoplasmic domain peptide(such as one with SEQ ID NO:45, or any of SEQ ID NOs: 5-44) than theaffinity or selectivity of the binding entity for a full-length gEcytoplasmic domain (e.g. with SEQ ID NO:1 or 4), or vice versa. Forexample, binding entities can be employed in the methods, compositions,kits and devices described herein that bind to gE cytoplasmic tailpeptide (such as one with any of SEQ ID NOs:5-45) with at least 50% orgreater affinity (or selectivity), or 60% greater affinity (orselectivity), or 70% greater affinity (or selectivity), or 80% greateraffinity (or selectivity), or 85% greater affinity (or selectivity), or90% greater affinity (or selectivity), or 95% greater affinity (orselectivity) than to a gC cytoplasmic tail domain with SEQ ID NO:1 or 4.

Similarly, for example, a binding entities can be employed in themethods, compositions, kits and devices described herein that bind withgreater affinity or selectivity to a gE cytoplasmic tail domain with SEQID NO:1 or 4, than to a peptide with any of SEQ ID NO:5-45. For example,the binding entity can bind to gE cytoplasmic tail domain with SEQ IDNO:1 or 4 with at least 50% or greater affinity (or selectivity), or 60%greater affinity (or selectivity), or 70% greater affinity (orselectivity), or 80% greater affinity (or selectivity), or 85% greateraffinity (or selectivity), or 90% greater affinity (or selectivity), or95% greater affinity (or selectivity) than the binding entity binds to apeptide any of SEQ ID NO:5-45.

Binding entities can be separated from impurities before incorporationinto the compositions, kits or devices. For example, the bindingentities can be purified or isolated using purification methods such aselectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing, and the like.The degree of purification necessary will vary depending on thecontaminants present with the binding entities. In some instances nopurification will be necessary (e.g., when binding entities arecommercially available and provided in purified form).

Antibodies directed against gE cytoplasmic domain with SEQ ID NO:1 or 4;or against a gE cytoplasmic domain peptide with any of SEQ ID NO:5-45are often monoclonal antibodies.

A monoclonal antibody is a population of molecules having a commonantigen binding site that binds specifically with a particular antigenicepitope. A monoclonal antibody can be obtained by selecting anantibody-producing cell from a mammal that has been immunized with aselected antigen such as a gE cytoplasmic domain with SEQ ID NO:1 or 4,or a peptide thereof with any of SEQ ID NO:5-45, and fusing theantibody-producing cell, e.g. a B cell, with a myeloma to generate anantibody-producing hybridoma. A monoclonal antibody can also be obtainedby screening a recombinant combinatorial library such as an antibodyphage display library. See, for example, PHAGE DISPLAY—A LABORATORYMANUAL, Barbas, et al., eds. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; and Kontermann & Dëbel, ANTIBODY ENGINEERING,Heidelberg: Springer-Verlag. Berlin, 2001. Techniques for preparingmonoclonal antibody-secreting hybridoma cells are also described, forexample, by Kohler and Milstein, Nature 256:495-97 (1975) and Kozbor etal. Immunol Today 4: 72 (1983).

A monoclonal antibody against a gE cytoplasmic domain or peptide thereofcan also be prepared using other methods available in the art. Forexample, the antibodies can be obtained by screening of a recombinantcombinatorial immunoglobulin library using a selected gE cytoplasmicdomain or peptide thereof (e.g., any of SEQ ID NO:1, 4, 5-44 or 45)Immunoglobulins that selectively bind to a selected gE cytoplasmicdomain or peptide thereof can be produced by recombinant expression fromcells encoding the immunoglobulin of interest.

The antibodies can be evaluated for affinity to a selected against gEcytoplasmic domain or peptide thereof using standard proceduresincluding, for example, enzyme linked immunosorbent assay (ELISA) todetermine antibody titer, and protein A chromatography to obtain theantibody-containing an IgG fraction.

Another method for generating antibodies involves a Selected LymphocyteAntibody Method (SLAM). The SLAM technology permits the generation,isolation and manipulation of monoclonal antibodies without needing togenerate a hybridoma. The methodology principally involves the growth ofantibody forming cells, the physical selection of specifically selectedantibody forming cells, the isolation of the genes encoding the antibodyand the subsequent cloning and expression of those genes.

The nucleic acids encoding the antibodies can be mutated to optimize theaffinity, selectivity, binding strength or other desirable property ofan antibody. A mutant antibody refers to an amino acid sequence variantof an antibody. In general, one or more of the amino acid residues inthe mutant antibody is different from what is present in the referenceantibody. Such mutant antibodies necessarily have less than 100%sequence identity or similarity with the reference amino acid sequence.In general, mutant antibodies have at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the reference antibody. Preferably,mutant antibodies have at least 80%, more preferably at least 85%, evenmore preferably at least 90%, and most preferably at least 95% aminoacid sequence identity or similarity with the amino acid sequence ofeither the heavy or light chain variable domain of the referenceantibody.

Labels

A variety of different labels can be used in the methods, compositions,binding entities, kits, and devices described herein. Labels can becovalently attached to any of the binding entities, primers or probesdescribed herein. Alternatively, the labels can non-covalently associatewith a hybridized probe or primer that is specifically bound to a targetnucleic acid (e.g., an mRNA encoding a gE cytoplasmic domain or peptidethereof). Similarly, a label can be non-covalently or indirectly boundto a binding entity. For example, the label can be an enzyme substratethat changes into a detectable (e.g., colored) product when exposed toan enzyme. Alternatively, the label can be an enzyme that generates acolored signal when exposed to a substrate.

So called “direct labels” are detectable labels that are directlyattached to or incorporated into a binding entity that then can bind toan gE cytoplasmic domain or peptide thereof. In contrast, so-called“indirect labels” are joined to a complex formed between a gEcytoplasmic domain or peptide thereof, and a binding entity aftercomplex formation. For example, an indirect label can be attached to asecondary antibody that binds to a different epitope on a gE cytoplasmicdomain or peptide thereof, than does a primary antibody. In anotherexample, the label can be attached to a secondary antibody that binds toa primary antibody that is already bound to a gE cytoplasmic domain orpeptide thereof.

Examples of labels include, but not limited to, fluorophores,chromophores, radiophores, enzymes, enzymatic substrates, enzymatictags, antibodies, chemiluminescence, electroluminescence, and affinitylabels. One of skill in the art will recognize that these and otherlabels can be used with success in this invention. Examples of enzymelabels include enzymes such as urease, alkaline phosphatase, orperoxidase to mention a few. Colorimetric indicator substrates can beemployed to provide a detection means visible to the human eye orspectrophotometrically. Examples of fluorophores include, but are notlimited to, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665,BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3,Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green 500,Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red,ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red.

Means of detecting such labels are well known to those of skill in theart. For example, fluorescent markers may be detected using amicroscope, photodetector or fluorimeter to detect emitted light. Instill further examples, enzymatic labels can be detected by providingthe enzyme with a substrate and detecting the reaction product producedby the action of the enzyme on the substrate, and colorimetric labelsare detected by simply visualizing the colored label or reactionproduct; or by use of spectrometer.

Immunological Assay Device

A device for detecting BoHV-1 infection is shown in FIG. 7. FIG. 7Ashows a schematic diagram of a device with an elongated housing 10 thatcontains a lateral flow strip 20. The lateral flow strip 20 extendssubstantially the entire length of housing 10. The lateral flow strip 20is divided into a sample application area 40 positioned below a sampleintroduction port 30, an antigen-antibody conjugation site 50, a capturearea 60, and a distal absorbent pad 70. The antigen-antibody conjugationsite 50 can have mobile antigens 55 (e.g, a glycoprotein E cytoplasmictail or peptide thereof). The flow strip 20 can also have a backing 80.The mobile antigen 55 in the antigen-antibody conjugation site 50 can belabeled antigens (such as gold-conjugated antigen) that can react withand bind to antibodies in a test sample from an animal. A flow pathalong the lateral flow strip 20 passes from the sample application area40, through the antigen-antibody conjugation site 50, into the capturearea 60. Immobilized binding entities such as an antibody thatrecognizes the a constant region of bovine antibodies, are positioned oncapture area 60. The mobile antigens 55 can bind one or more antibodiesthat may be present in a test sample and the liquid flow can transport aconjugate formed between a mobile antigen and an antibody to the capturearea 60, where immobilized binding entities can capture theantigen-antibody conjugates and concentrate the label in the capturearea 60.

The mobile antigens 55 without a bound antibody pass through the capturearea 60 and are eventually collected in the distal absorbent pad 70.

The lateral flow strip 20 can also include a reaction verification orcontrol area 90. Such a control area 90 (e.g., configured as line) canbe slightly distal to the capture area 60. The reaction verification orcontrol area 90 illustrates to a user that the test has been performed.Prior to the test being performed, the reaction verification or controlarea 90 is not visible. However, when the test is performed by placing afluid sample on the sample application area 40, the reactionverification or control area 90 can become visible as the sample flowsthrough the capture area 60 and to the distal absorbent pad 70. Forexample, the reaction verification or control area 90 can become visibledue to a chemical reacting with any component of the sample or simplydue to the presence of moisture in the sample.

Quantitative results can be ascertained based on the magnitude of theidentifiable signal from the capture area 60 after application of asample to a sample introduction port 30. For example, in FIG. 7B asignal represented by a series of asterisks (*****) is present in thecapture area 60. The signal can be identified by visual inspection ofthe device, and/or the signal can be recorded and/or processed by adetector or a smart device. Such a smart device can be a small pocketcomputer, a laptop, a netbook, a desktop computer, or a smart phone. Forexample, the subject can display and/or record the results by taking aphotograph of the detection device after testing by use of a smartdevice. The smart device can be configured to interpret the results bydetecting the type and/or strength of a signal in the capture area 60 ofa detection device.

Such a lateral flow device can be small enough for transportation in apocket, mobile lab pack, backpack, or satchel. For example, lateral flowdevices can be about 1 to 3 inches long and about 0.25 to about 1.5inches wide. In general, the lateral flow devices are thin, having adepth of only about 0.1 to 0.75 inches.

Another device that can be used to detect BoHV-1 infection is adirect-charge transfer conductometric biosensor. Such direct-chargetransfer conductometric biosensors are similar in structure to lateralflow devices and can be provided in a package that is small, forexample, no larger than a pack of gum. Direct-charge transferconductometric biosensors can employ immobilized antigen and polyaniline(emeraldine salt) as a transducer for detection. The antigen can be aglycoprotein E cytoplasmic tail polypeptide or peptide thereof) that canbind to anti-BoHV-1 antibodies that may be present in a test sample.Electron charge flow is aided through conductive polyaniline, togenerate an electronic signal that can be recorded by a data collectionsystem.

The biosensor architecture can be similar to a lateral flow device.Polyaniline bound to mobile antigen can first capture anti-BoHV-1antibodies in a test sample, if present, to form a complex between themobile antigen molecules and the antibodies. The complex can flow bycapillary action to a capture site in the device where a complex canform with immobilized antibodies that bind to the anti-BoHV-1antibodies. Such interaction provides a direct electron charge flow togenerate a resistance signal that can be observed and/or recorded. Thedevice is easy to use, and provides fast but sensitive results. See,e.g., Pal et al., Biosens. Bioelectron. 22(9-10): 2329-36 (2007) (whichis incorporated herein by reference in its entirety).

For example, electrically active polyaniline coated magnetic (EAPM)particles (e.g., nanoparticles) can be synthesized by coating thesurface of gamma iron oxide cores with aniline monomers that areelectrically active. The aniline monomers can be made to be electricallyactive, for example, by acid doping. Antigen molecules are adsorbed orcovalently attached to the EAPM particles. The EAPM particles combinedwith the mobile antigen can be incorporated into a biosensor configuredsimilar to a lateral flow device. Capillary flow of the complex betweenantibodies from the test sample and the antigen-EAPM particles.Detection of the complex formed between the antibodies and theantigen-EAPM particles occurs as the complex flows into a capture regionwhere direct-charge transfer can occur across the EAPM particles.

Thus, to form this type of device, two or more electrodes can bescreen-printed onto a solid substrate (e.g., paper, nitrocellulose orother convenient substrate), where the distance between the electrodesis sufficient to isolate each electrode from another until chargetransfer occur across the EAPM particles in the capture region betweenthe electrodes. The surface of a solid support (e.g. a nitrocellulosemembrane) can be modified by a crosslinking agent such as glutaraldehydeto immobilize a binding entity (e.g., that reacts with antibodies in thetest sample) within a capture region of the biosensor. The mobileantigen EAPM particles with any bound antibodies are drawn by capillaryaction to the capture region. The polyaniline acts as an electric signaltransducer that signals binding between the mobile antigen-antibody EAPMparticles and the immobilized binding entities. Such an electricalresponse can be measured by pulse mode measurement, which provides aquantitative measure of the amount of anti-BoHV-1 antibodies bound tothe capture site.

For example, FIG. 7C is a schematic diagram of a schematic diagram of adirect-charge transfer conductometric biosensor with two electrodes 65and 67, that can transfer a charge across the EAPM particles in thecapture region 60 between the electrodes. FIG. 7D schematicallyillustrates collection of EAPM particles (*****) in the capture region60 between the electrodes.

Infection of BoHV-1 can be identified by visual inspection of the deviceand/or by the transfer of charge between the electrodes that provides anelectrical signal communicating whether or not BoHV-1 infection ispresent. The signal can also be transmitted via a transmitter 95 to areceiver or smart device (e.g., a small pocket computer, a laptop, anetbook, a desktop computer, or a smart phone), which can receive theresults, record the results, and alert personnel that a BoHV-1 infectionhas been detected.

Another detection device that can be employed to detect BoHV-1 infectionutilizes Radio Frequency Identification (RFID), where tags (“smartlabels”) provide a signal to a handheld or stationary reader. Such RFIDdetection devices can be provided in a package that is the approximatelythe same size as a pack of gum or smaller. The radio frequency tags canbe of various shapes, sizes, and readout ranges. For example, the tagscan be minute, and the detection strips with the tags can be thin andflexible. In some cases, radio frequency identification devices can havetags on a paper or plastic strip. Such RFID tags can contain siliconchips and, in some embodiments, antennas. Passive tags require nointernal power source, whereas active tags require an internal powersource.

The configuration of an RFID-containing BoHV-1 detection device can besimilar to a lateral flow device. For example, the RFID device can havea sample port into which a sample is loaded. Such a sample may comprise,for example, saliva, whole blood, plasma, serum, urine, lymph, etc. Thesample can flow into a antigen-antibody conjugation site whereantibodies in a test sample can bind to mobile RFID-labeled antigens.The sample passes (e.g., by capillary flow) into an RFID capture andidentification zone, in which the RFID-antigen-antibody complexes areimmobilized, for example, by binding to an immobilized binding entitythat binds to bovine antibodies (e.g., to the constant region of bovineantibodies). The capture region is then subjected to RFID interrogationby an RFID detector. The unreacted RFID-labeled antigens (i.e., notbound to an antibody from the test sample) and other components of thesample pass through the RFID capture and identification zone into aradio frequency protected “waste” zone, which can have shielding toprevent RFID tags therein from being detected by the RFID detector. Thedetection of a particular RFID tag by the RFID detector is indicativethat anti-BoHV-1 antibodies are present in the sample. See, e.g., US20120269728 by Jen et al., which is specifically incorporated herein byreference in its entirety.

FIG. 7E is a schematic diagram illustrating a detection device that hasRFID tags as labels on antigens 55. The device can have an unshieldedantigen-antibody conjugation site 50, and shield 75 over distalabsorbent pad 70, so that collection of RFID tags in the capture area 60can transmit a radio signal (represented by *****, see FIG. 7F). Such asignal indicates that one anti-BoHV-1 antibodies are present in the testsample and have been detected by the device.

A detector or reader can receive a passive or active tag signal from theRFID test strip. A Passive Reader Active Tag (PRAT) system has a passivereader that only receives radio signals from active tags (batteryoperated, transmit only). The reception range of a PRAT system readercan be adjusted from 1-2,000 feet, allowing some flexibility in thefield. The detector or reader can be a smart device.

An Active Reader Passive Tag (ARPT) system has an active reader, whichtransmits interrogator signals and also receives authentication repliesfrom passive tags. An Active Reader Active Tag (ARAT) system uses activetags awoken with an interrogator signal from the active reader. Avariation of this system can involve use of a Battery Assisted Passive(BAP) tag which acts like a passive tag but has a small battery to powerthe tag's return reporting signal. The detector or reader can be a smartdevice, which can receive radio signals via Bluetooth transmissionand/or reception.

Another device that can be used for determining the presence of BoHV-1infection in a fluid sample is a test strip. The test strips can beprovided in a roll, where a section of the roll is torn off for testing.Alternatively, the test strips can be provided in a package that is theapproximately the same size as a pack of gum or smaller. The test stripcan have one or more gE CT antigens (e.g., one or more with SEQ IDNOs:1, 4-44, and/or 45) immobilized thereon. A bodily fluid can beapplied to the test strip and a signal can be detected when anti-BoHV-1antibodies are present in the bodily fluid. To enhance the signal, thestrip can optionally be placed in a development solution. The signalindicates that the sample is from an animal infected with BoHV-1.

FIG. 8 shows an example of a roll of test strips 100. The roll of teststrips 100 has one or more types of immobilized antigens 110 that canspecifically bind to anti-BoHV-1 antibodies that may be present in atest sample. A subject can tear a test strip segment 105 from the rollof test strips 100. The test sample (e.g. a bodily fluid such as serum,blood, saliva, mucus, or urine) can be applied anywhere along the teststrip segment 105. A signal can be detected from the test strip when atest sample contains antibodies against BoHV-1. A rinsing liquid 120(e.g., water or PBS) can be used to remove unbound materials after thesample has been applied to the test strip segment 105. The test stripsegment 105 can optionally be immersed in one or more developmentsolutions 130 that can be provided with roll of test strips 100. Thesignal can directly be detected or the strip can optionally be rinsed ina rinsing liquid 140 (e.g., water or PBS). The signal (e.g. a color)indicates that the sample is from an animal infected with BoHV-1. Theroll test strip 100 can be elongated rectangular material wound into aroll. Segments 105 of the roll of test strips 100 are easily removed ortorn from the roll without loss or changes in the detection propertiesof the materials that make up the roll of test strips 100. In certainexamples, the roll of test strips 100 can be composed of paper,nitrocellulose, or other porous inert materials. The test strips canalso be in the form of short strips (e.g., about 1 to 3 inches long),that are provided in a package that is the approximately the same sizeas a pack of gum or smaller.

The test strips can be flexible and readily transported as individualstrips in a small package, or as a continuous roll of test stripmaterial. The test strip can vary in width, thickness, and length. Forexample the test strip can be about 0.2 to about 1 inch wide. The teststrip can be as thick as a piece of paper or somewhat thicker, forexample, about 0.02 to about 0.5 inches thick. Individual strips can beabout 1 inch to 3 inches long. A continuous roll test strip material canbe of varying length, for example, a length that allows easytransportation in a pocket or handbag when rolled Up.

The development solution(s) can contain reagents that recognize theantigen-antibody complex and that provide a visually detectable signalidentifying that such a complex has formed. For example, a developmentsolution can contain a secondary binding entity or antibody that bindsto anti-BoHV-1 antibodies to form a ternary complex with anantigen-antibody immobilized on the test strip. The secondary bindingentity or antibody can be linked to a visually detectable label, or itcan be linked to an entity that can generate a visually detectablesignal upon exposure to a substrate. For example, the secondary bindingentity or antibody can be linked to an enzyme to produces a visuallydetectable signal upon exposure to a substrate for the enzyme.

Quantitative results can be ascertained based on the magnitude of theidentifiable signal from the test strip segment 105 that has beentreated as described above. A smart device can be used to display,record, graph, and/or interpret the results. For example, the methodsdescribed herein can include a step of photographing the results shownon the detection device using a smart device. The smart device canrecord, store, process, and display the results as well as graphicinterpretations of the results.

Hence a variety of devices can be used to evaluate test samples forBoHV-1 infection.

Kits

To provide those skilled in the art with tools to use the presentinvention, the glycoprotein E cytoplasmic tail polypeptide or peptide(e.g., any polypeptide or peptide with SEQ ID NOs: 1, 4-44, 45, or acombination thereof) can be assembled into kits for the diagnosis,detection or confirmation of BoHV-1. The presence of antibodies reactiveto glycoprotein E cytoplasmic tail polypeptide or peptide thereof (e.g.,any polypeptide or peptide with SEQ ID NOs: 1, 4-44, 45, or acombination thereof) is used to identify animals infected with BoHV-1and/or animals that would benefit from vaccination with the BoHV-1 tmvvaccine. The information provided is also used to direct the course oftreatment. For example, if a subject is found to have antibodies againstto glycoprotein E cytoplasmic tail polypeptides (e.g., SEQ ID NO:1 or 4)or peptides thereof, (e.g., any of SEQ ID NOs: 5-45) provide usefulantigens for detection of antibodies that are circulating in BoHV-1animals.

The kits can include a carrier means for the devices and reagents aswell as other components of the kits. Such a carrier can be a box, abag, a satchel, plastic carton (such as molded plastic or other clearpackaging), wrapper (such as, a sealed or sealable plastic, paper, ormetallic wrapper), or other container. In some examples, kit componentswill be enclosed in a single packaging unit, such as a box or othercontainer, which packaging unit may have compartments into which one ormore components of the kit can be placed. In other examples, a kitincludes one or more containers, for instance vials, tubes, and the likethat can separately contain, for example, one or nucleic acid probes,one or more binding entities, one or more devices, as well as positiveand/or negative control samples or solutions.

For example, at least one of the containers can include at least BoHV-1antigen such as a glycoprotein E cytoplasmic tail polypeptide (e.g., SEQID NO:1 or 4) or a peptide thereof (e.g., any of SEQ ID NOs: 5-45). Inanother embodiment, at least one of the containers can include at leastone binding entity that binds with specificity or selectivity to theBoHV-1 glycoprotein E cytoplasmic tail polypeptide (e.g., SEQ ID NO:1 or4) or a peptide thereof (e.g., any of SEQ ID NOs: 5-45). The bindingentities, can be detectably labeled. For example, the binding entitiescan be packaged separately from the labels, and the label can be addedto the binding entities, during or after performance of an assay fordetecting BoHV-1.

Kits can also contain vials, needles, syringes, finger-prick devices,alcohol swabs, gauze squares, cotton balls, bandages, latex gloves,incubation trays with variable numbers of troughs, adhesive platesealers, data reporting sheets, which may be useful for handling,collecting and/or processing biological samples. Kits may alsooptionally contain implements useful for introducing samples into anassay chamber or a cell capturing device, including, for example,droppers, Dispo-pipettes, capillary tubes, rubber bulbs (e.g., forcapillary tubes), and the like. Other components can also be present inthe kits such as disposal means for discarding used devices and/or otheritems used with the device (such as patient samples, etc.). Suchdisposal means can include, without limitation, containers that arecapable of containing leakage from discarded materials, such as plastic,metal or other impermeable bags, boxes or containers.

The kits can include instructions for performing an assay such as animmunoassay.

Use of the methods, compositions, devices, and kits described hereinfacilitate early detection of BoHV-1 infection, and identify animalsthat should be vaccinated and/or treated for infection.

The following non-limited Examples illustrate some of the materials,methods, and experiments used in the development of the invention.

Example 1: Materials and Methods

This Example describes some of the materials and methods used indeveloping the invention.

Cells and Virus Strain.

The Madin-Darby bovine kidney (MDBK) cell line obtained from theAmerican Type Culture Collection (Manassas, Va.) was maintained inDulbecco's modified Eagle's medium (DMEM) supplemented with 5-10%heat-inactivated fetal bovine serum (FBS) (HyClone Laboratories, Inc.,South Logan, Utah). The BHV-1 Cooper (Colorado-1) strain, obtained fromthe American Type Culture Collection (Cat # CRL-1390; Manassas, Va.),was propagated and titrated in MDBK cells as described by Chowdhury(Microbiol 52(1-2):13-23 (1996)).

The BoHV-1 tmv virus containing deletions of UL49.5 residues 30-32 and80-96, gE cytoplasmic tail (CT) residues 452-575, and the entire Us9gene, as well as the BoHV-1 gE-deleted virus with a deletion of theentire gE open reading frame, were constructed as described by Wei &Chowdhury, PloS one 6(10):e25742 (2011); and Brum et al. J Neurovirol15(2):196-201 (2009)). The BoHV-1 Cooper (Colorado-1) strain (ATCC Cat #CRL-1390, Manassas, Va.) was used as the reference wild type strain.

Monoclonal/Polyclonal Antibodies.

Mouse anti His-tag MAb (Genscript Cat # A00186) and/or goat anti gECT-specific antibody (Chowdhury et al., Vaccine 32(39):4909-4915 (2014))goat anti mouse-HRP conjugated polyclonal antibody (Thermo Scientific)were used to confirm reactivity and specificity of the recombinantprotein and MAb respectively.

Construction of a gE CT Domain-Expressing pET-43.1a Plasmid.

The amino acid sequence of residues 451-575 of the gE cytoplasmic tail(CT) is shown in FIG. 1B (SEQ ID NO:1). An E. coli codon-optimizednucleic acid segment (SEQ ID NO: 2) was synthesized that encodes the SEQID NO:1 gE CT polypeptide domain (FIG. 1C). This SEQ ID NO:2 nucleicacid segment was modified to generate the SEQ ID NO:3 nucleic acid thatincludes, at the 5′ end, a Nde I restriction site followed by amethionine codon and six histidine codons. At the 3′ end, the SEQ IDNO:3 construct has a stop codon followed by Xhol restriction site (FIG.1D). Hence, the SEQ ID NO:3 nucleic acid encodes the modified gE CT-Hispolypeptide (SEQ ID NO:4) shown in FIG. 1E. The 411 bp long Ndel/Xholfragment (SEQ ID NO:3) was inserted into the Ndel-Xhol sites ofpET-43.1a (Novagen) to create the pET-43.1a gE CT-His construct. Themolecular weight of the E. coli expressed and purified gE CT-His wasverified by SDS-PAGE.

Production and Characterization of gE CT-Specific Mouse MonoclonalAntibody.

The gE CT-His protein (SEQ ID NO:4) was expressed from the pET-43.1a gECT-His construct within E. coli. Protein purification, immunization ofmice, production of hybridomas, purification of the MAb 2H8F3 from theascites fluid and conjugation of the MAb with HRP (horse reddishperoxidase) were performed by Genscript. Purification of gE CT-His fromE. coli, immunoblotting analysis and generation of mouse monoclonalantibody (MAb).

The E. coli expressed gE CT-His protein (SEQ ID NO:4) was obtained frominclusion bodies, solubilized with 8M urea and purified in two steps byuse of a Ni column and Superdex 200 (GE Healthcare). E. coli-expressedgE CT-His protein thus purified, was then tested for reactivity withanti-His antibody (Genscript cat. # A00186). Mice were immunized withthe purified gE CT-His protein. Anti-His negative, gE CT-specifichybridomas were selected for further processing. Hybridomas producing gECT-specific antibodies were selected for the production of mouseascites. The 2H8F3 monoclonal antibody was thereby generated.

Western Blot Analysis of Recombinant gE CT-His Protein with gECT-specific MAb.

The gE CT-His antigen protein samples were separated on a SDS-PAGE (10%or 12%) and transferred onto a nitrocellulose membrane. The membrane wasblocked in 5% skimmed milk in PBS and/or TBS for 1 hr at roomtemperature (RT), washed with 0.05% tween 20 in PBS (PBST) or TBS (TBST)and incubated with antibody (0.5 μg/ml diluted in PBS or TBS for 2-3hours or overnight at room temperature). After washing, the membrane wasincubated either with secondary antibody goat anti mouse HRP or withsecondary antibody IRDye800CW goat anti-mouse IgG at room temperaturefor 1 hour. After washing, the membrane was visualized either withPierce ECL western blotting substrate/autoradiography or under Odysseyscanner (LI-COR, Odyssey V3.0).

Synthesis of Biotinylated, Overlapping Peptides, and Epitope MappingELISA.

Thirty nine 12-mer overlapping peptides (Table 1) spanning the entire125 aa residues of gE CT (FIG. 1A-1B) were synthesized and biotinylated(Genscript).

TABLE 1  Peptides for Epitope Mapping SEQ ID No. Peptide Sequence NO:  1ASQKRTYDILNP  5  2 KRTYDILNPFGP  6  3 YDILNPFGPVYT  7  4 LNPFGPVYTSLP  8 5 FGPVYTSLPTNE  9  6 VYTSLPTNEPLD 10  7 SLPTNEPLDVVV 11  8 TNEPLDVVVPVS12  9 PLDVVVPVSDDE 13 10 VVVPVSDDEFSL 14 11 PVSDDEFSLDED 15 12DDEFSLDEDSFA 16 13 FSLDEDSFADDD 17 14 DEDSFADDDSDD 18 15 SFADDDSDDDGP 1916 DDDSDDDGPASN 20 17 SDDDGPASNPPA 21 18 DGPASNPPADAY 22 19 ASNPPADAYDLA23 20 PPADAYDLAGAP 24 21 DAYDLAGAPEPT 25 22 DLAGAPEPTSGF 26 23GAPEPTSGFARA 27 24 EPTSGFARAPAN 28 25 SGFARAPANGTR 29 26 ARAPANGTRSSR 3027 PANGTRSSRSGF 31 28 GTRSSRSGFKVW 32 29 SSRSGFKVWFRD 33 30 SGFKVWFRDPLE34 31 KVWFRDPLEDDA 35 32 FRDPLEDDAAPA 36 33 PLEDDAAPARTP 37 34DDAAPARTPAAP 38 35 APARTPAAPDYT 39 36 RTPAAPDYTVVA 40 37 AAPDYTVVAARL 4138 DYTVVAARLKSI 42 39 TVVAARLKSILR 43

The biotinylated peptides (20 μg/ml) were immobilized onto 96-wellmicrotiter plates pre-coated with 100 μl of 10 μg/ml streptavidin (NEB,Cat # A00160). In addition, a microtiter plate was coated with purifiedantigen protein gE CT-His (2 μg/ml) in 100 μl coating buffer at 4° C.overnight. The plates were then blocked with the blocking buffer (5%skimmed milk in PBS) at 37° C. for 2 hours. After washing the plate withwashing buffer (PBS with 0.05% tween), 100 μl of 1 μg/ml MAb 2H8F3 wasadded to the plates and incubated at 37° C. for 2 hours. The plates werewashed with washing buffer (PBST) and then incubated with 100 μl ofsecondary antibody (0.1 μg/ml goat anti-mouse IgG [HRP]) at 37° C. for 1hour. After washing, the reaction was developed with 100 μl TMBsubstrate for 10 minutes at room temperature. The reaction was stoppedby adding 100 μl of 1 M HCl. The absorbance of each well was measured at450 nm using a spectrometer. An uncoated well was used as a blankcontrol. The peptide library incubated with the secondary antibody onlywas also used as a negative control.

Infection of Calves.

Animal infection, handling, sample collection and euthanasia protocolswere previously approved by the LSU Institutional Animal Care and UseCommittee. To determine the differential serological marker propertiesof BoHV-tmv in infected/vaccinated calves compared with the BoHV-1gE-deleted and BoHV-1 wt-infected calves, twelve BoHV-1 and BVDVnegative, 4-month-old cross-bred bull calves were selected and randomlyassigned into three groups. The first group consisted of three calvesinfected with wild type BoHV-1. The second group consisted of fourcalves immunized with the BoHV-1 gE-deleted vaccine. The third groupconsisted of five calves immunized with the BoHV-1 tmv vaccine. Calvesin each group were housed at the LSU large animal isolation facilitywell isolated from each other in separate rooms, where 2-3 calves weremaintained per room. Calves in each group were infected intranasallywith 1×10⁷ PFUs per nostril of the respective virus or vaccine. Thus,each calve received 2×10⁷ PFUs of virus/vaccine. Following infection,nasal swabs were collected every other day until day 10 post-infection.Sera samples were collected at days 0, and 28 days post-infection, thenaliquoted and stored at −80° C. until further analysis.

Competitive ELISA Tests.

Competitive ELISA was performed to detect the presence or absence of gECT antibodies among the three groups of calve sera: wild type (WT)BoHV-1 infected calve serum, BoHV-1 tmv vaccinated calve serum, and gEdeleted (gEA) vaccinated calve serum. This was accomplished bycollecting samples of WT, BoHV-1 tmv, and gEΔ calf sera at day 0 (priorto infection) and 28 days post infection (dpi), then testing for thepresence of anti-gE antibodies.

An ELISA plate (Costar, #3590) was coated overnight at 4° C. with 200μl/well (approx. 100 ng) of gE CT-His protein antigen in a coatingbuffer (30 mM Na₂CO₃/70 mM NaHCO₃ pH9.6). After washing with 0.05% Tween20-PBS pH 7.4 (PBST) followed by 1×PBS pH 7.4 (PBS), the plates wereblocked with 1% bovine serum albumin (BSA) in PBS at 37° C. for 2 hours.After washing, the plates were incubated overnight at 4° C. with 100 μlof calf sera diluted 1:1 in 0.1% PBST. The next day, the plates werewashed and incubated for 1 hour at room temperature with 100 μ1 of HRPconjugated anti-BoHV-1 gE CT-specific Mab 2H8F3 antibodies diluted1:10,000 in 0.05% PBST/well. Plates were washed and 150 μl of TMBsubstrate was added to each well. The plates were incubated at roomtemperature in the dark for 15 minutes, and then 75 μl of 1M H₂SO₄ wasadded to stop the reaction. The optical density (OD) of each well wasread at 450 nm using a microtiter plate reader (SpectraMax M2, MolecularDevices with Soft Max Pro 4.8).

Statistical Analysis: The SAS® (version 9.4, SAS Institute, Cary, N.C.)mixed procedure was used to analyze the competitive ELISA data with arepeated measures analysis of variance in a mixed effects model. Fixedeffects included Sample, DPI and the interaction Sample*DPI. The randomeffect in the model was Calf (Sample). When overall differences werefound, post hoc comparisons were made with pairwise “t” tests ofleast-squares means. All comparisons were considered significant atp≤0.05.

Example 2: gE Cytoplasmic Tail-Specific Antibody Distinguishes Wild TypeBoHV-1 Virus-Infected from BoHV-1 Tmv-Infected cells

As shown in FIG. 1E, the molecular weight of the E. coli expressedrecombinant gE CT-His protein is predicted to be 14.3 kD using thecalculator provided by ExPASy.org Immunoblotting experiments with theanti-His antibody indicated that gE CT-His protein expressed in E. colifrom the pET-43.1a gE CT-His construct had a slightly higher molecularweight than the predicted 14.3 kD (FIG. 2A). Therefore, MALIDI-TOF massspec analysis was performed to assess the actual mass of the expressedprotein. Such analysis showed that the molecular weight of the expressedgE CT-His protein was very similar as the predicted molecular weight(FIG. 3).

Subsequently, a BoHV-1 gE CT-specific mouse monoclonal antibody(MAb2H8F3) was generated using Genscript technology by immunizing micewith the gE CT-His protein as antigen. Further immunoblotting analysisdemonstrated that the same E. coli-expressed gE CT-His protein shown inFIG. 2A-B is recognized by MAb2H8F3 (FIG. 2C) but not by an antiGST-specific antibody (FIG. 2D).

Additionally, the MAb2H8F3 antibody reacted specifically with a 94 kDband corresponding to BoHV-1 wild type gE that had been expressed bywild type BoHV-1 virus-infected cells. However, the MAb2H8F3 antibodydid not react with a 68 kD band expressed by BoHV-1 tmv-infected cells(FIG. 2E) because the BoHV-1 tmv vaccine lacks the gE CT domain.

These results demonstrate that the MAb2H8F3 antibody specificallyrecognizes an epitope located within the 125 amino acids of the gEcytoplasmic tail. More significantly, these results also indicate thatBoHV-1 tmv-infected cells can be distinguished from wild type BoHV-1virus-infected cells, for example, by using the MAb2H8F3 antibody todetect wild type BoHV-1 or by using the gE cytoplasmic tail as anantigen to detect antibodies that may be in the serum of BoHV-1virus-infected animals.

Example 3: Epitope Recognized by the MAb2H8F3 Antibody

To map the epitope that the specified by the MAb2H8F3 specificallyrecognizes in the BoHV-1 gE CT domain, thirty nine overlapping 12-merpeptides (Table 1) spanning the entire 125 amino acid cytoplasmic tailof the gE protein (SEQ ID NO:1) were generated and analyzed for bindingwith the gE CT-specific MAb2H8F3 antibody.

The ELISA was performed with plates where each well was coated with aseparate 12-mer peptide. One plate was reacted with the gE CT-specificMAb2H8F3 antibody. A second plate with wells coated by the peptide ofthe library was incubated with the secondary antibody only as a negativecontrol. In addition, well number 40 contained streptavidin (no peptide)as blank control and well number 41 contained the E. coli expressedrecombinant gE CT-His polypeptide (SEQ ID NO:4).

The ELISA test results presented in Table 2 and FIG. 3 show that theMAb2H8F3 reacted specifically with the two overlapping peptides:

Peptide₄₉₆DDSDDDGPASN₅₀₇ (SEQ ID NO:20; peptide 16 in Table 1) and

Peptide₄₉₉SDDDGPASNPPA₅₁₀(SEQ I DNO:21; peptide 17 in Table 1).

Therefore, the epitope bound by the MAb2H8F3 antibody maps within gEamino acid positions 499-507.

TABLE 2 ELISA results of epitope mapping for MAb 2H8F3. Secondary AbPeptide/Well MAb2H8F3 (only) No. SEQ ID NO: O.D. 450 nm O.D. 450 nm 1 50.152 0.062 2 6 0.096 0.074 3 7 0.093 0.058 4 8 0.097 0.058 5 9 0.0890.056 6 10 0.097 0.063 7 11 0.099 0.060 8 12 0.103 0.059 9 13 0.0830.058 10 14 0.101 0.061 11 15 0.094 0.059 12 16 0.095 0.063 13 17 0.0820.058 14 18 0.095 0.060 15 19 0.087 0.052 16 20 0.450 0.056 17 21 0.6550.053 18 22 0.082 0.053 19 23 0.087 0.052 20 24 0.102 0.054 21 25 0.0980.053 22 26 0.086 0.051 23 27 0.101 0.055 24 28 0.098 0.059 25 29 0.1050.060 26 30 0.097 0.055 27 31 0.121 0.053 28 32 0.100 0.054 29 33 0.0980.056 30 34 0.087 0.051 31 35 0.081 0.052 32 36 0.089 0.056 33 37 0.0660.056 34 38 0.097 0.052 35 39 0.089 0.055 36 40 0.111 0.066 37 41 0.0940.058 38 42 0.099 0.054 39 43 0.096 0.053 40 — 0.051 0.055 41 4 2.3590.057

Analysis of the two peptide sequences (SEQ ID NOs: 20-21) demonstratethat the nine amino acids ₄₉₉SDDDGPASN₅₀₇ (SEQ ID NO: 44) are commonbetween the two overlapping peptides. Based on these results the epitoperecognized by the MAb2H8F3 antibody within the gE CT domain is₄₉₉ESDDDGPASN₅₀₇ (SEQ ID NO:45). The MAb2H8F3 antibody is referred toherein as the HRP-conjugated indicator marker antibody

Example 4: Serological Assay for Distinguishing BoHV-1 WT-InfectedCalves from BoHV-1 tmv and/or gE-Deleted Virus-Infected Calves

The direct ELISA test described above was optimized using a series ofcoating antigen concentrations and HRP-conjugated MAb2H8F3 dilutions.Based on these analyses (data not shown), the optimal amount of gECT-His coating antigen and HRP-conjugated MAb 2H8F3 (indicator markerantibody) were selected to be 100 ng of gE CT-His antigen and 1:10,000dilution of the HRP conjugated MAb, respectively.

The competitive ELISA method was performed as follows. Inactivated calvesera obtained prior to infection (day 0) and at 28 days post-infectionwere incubated in separate microtiter wells coated with the gE CT-Hisantigen. After such incubation, the HRP conjugated indicator markerantibody was added and the ELISA plates were again incubated. The wellswere then washed, and the optical density at 450 nm of each sample wellwas determined. Inactivated, non-infected fetal calf serum (availablecommercially, Atlas biologicals), and hyper immune BoHV-1 wt-specificcalf serum (BoHV-1 HI) produced by multiple buster intermuscularimmunizations using purified BoHV-1 as antigen) were used as negativeand positive controls, respectively.

The measured optical density of test samples and controls at 450 nm wasused to calculate either a sample/negative control (S/N) ratio or apercent inhibition using the following algorithms:

${{Percent}\mspace{14mu}{inhibition}} = {\left( \frac{{A\; 450\mspace{14mu}{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} - {A\; 450\mspace{14mu}{OD}\mspace{14mu}{sample}}}{A\; 450\mspace{14mu}{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right) \times 100}$$\mspace{20mu}{{S\text{/}N\mspace{14mu}{ratio}} = \left( \frac{A\; 450\mspace{14mu}{OD}\mspace{14mu}{sample}}{A\; 450\mspace{20mu}{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right)}$If the S/N ratio was less than or equal to 0.6, the sample wasclassified as positive for the presence of anti-BoHV-1 antibodies (i.e.,the animal from which the serum was obtained was infected with BoHV-1).If the S/N ratio was greater than 0.7, the sample was classified asnegative for anti-BoHV-1 antibodies (i.e., the animal from which theserum was obtained was not infected with BoHV-1). If the SN ratio wasbetween 0.6-0.7, then the sample was classified as suspect.Results

The results of the competitive ELISA are summarized in Table 4, whichshows the assessed competitive/blocking ELISA results of calf serumsamples prior to infection (0 day) or at 28 days post infection (28 day)where the sera were obtained from BoHV-1 wt, BoHV-1 gEΔ and BoHV-1 tmvinfected/vaccinated calves. The S/N ratio and % inhibition values shownin Table 4 were calculated from mean O.D of three replicate ELISA testsfor each serum samples are shown.

TABLE 4 Serological Competitive ELISA Results Pos- Groups Samples %Inhibition S/N Ratio itive? Neg. Control FBS 0 1 N Pos. ControlBoHV-1-HI 71.02 0.290 Y 0 Day 28 Day 0 Day 28 Day BoHV-1 2309 10.2944.88 0.897 0.551 Y WT 2349 19.21 41.29 0.808 0.587 Y 2559 19.66 29.660.803 0.703 Sus- pect BoHV-1 26 15.84 27.30 0.842 0.727 N gEΔ 27 13.8531.48 0.862 0.685 Sus- pect 28 6.49 18.99 0.935 0.810 N 32 1.47 15.490.985 0.845 N BoHV-1 31 3.93 8.72 0.961 0.913 N TMV 33 9.35 10.16 0.9060.898 N 369 2.00 14.97 0.980 0.850 N 379 13.40 15.40 0.866 0.846 N 38510.16 10.96 0.898 0.890 N

In these competitive ELISA tests, day 0 sera obtained prior to infectionfrom all three calves were negative for anti-BoHV-1 antibodies (SNratio >0.7). Two of the three BoHV-1 wt infected calves were positivefor anti-BoHV-1 antibodies (SN ratio<0.6). However, sera from one BoHV-1wt infected calf had a somewhat higher SN ratio (SN ratio 0.7) (Table4).

The ELISA test results also showed that three out of four calvesinfected with the commercially available gE-deleted virus testednegative at 28 dpi while one had a slightly lower SN ratio (0.685).

However, all five calves infected with BoHV-1 tmv tested negative in thecompetitive ELISA at both day 0 and 28 days post-infection.

Statistical analysis revealed that there was an overall significantinteraction for Sample*DPI (p=0.021) with respect to SN Ratio. Post hoccomparisons indicated that there were significant differences on day 28post-infection between the BoHV-1 wt-infected versus BoHV-1 gE(p=0.0006) and BoHV-1 tmv (p=0.0025) viruses-infected calves.

REFERENCES

-   1. Jones C, Chowdhury S: Bovine herpesvirus type 1 (BHV-1) is an    important cofactor in the bovine respiratory disease complex. Vet    Clin North Am Food Anim Pract, 26(2):303-321.-   2. Jones C, Chowdhury S: A review of the biology of bovine    herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine    respiratory disease complex and development of improved vaccines.    Anim Health Res Rev 2007, 8(2):187-205.-   3. Koppers-Lalic D, Reits E A, Ressing M E, Lipinska A D, Abele R,    Koch J, Marcondes Rezende M, Admiraal P, van Leeuwen D,    Bienkowska-Szewczyk K et al: Varicelloviruses avoid T cell    recognition by UL49.5-mediated inactivation of the transporter    associated with antigen processing. Proc Natl Acad Sci USA 2005,    102(14):5144-5149.-   4. Nataraj C, Eidmann S, Hariharan M J, Sur J H, Perry G A,    Srikumaran S: Bovine herpesvirus 1 downregulates the expression of    bovine MHC class I molecules. Viral Immunol 1997, 10(1):21-34.-   5. Chowdhury S I, Wei H, Weiss M, Pannhorst K, Paulsen D B: A triple    gene mutant of BoHV-1 administered intranasally is significantly    more efficacious than a BoHV-1 glycoprotein E-deleted virus against    a virulent BoHV-1 challenge. Vaccine 2014, 32(39):4909-4915.-   6. Butchi N B, Jones C, Perez S, Doster A, Chowdhury S I: Envelope    protein Us9 is required for the anterograde transport of bovine    herpesvirus type 1 from trigeminal ganglia to nose and eye upon    reactivation. J Neurovirol 2007, 13(4):384-388.-   7. Liu Z F, Brum M C, Doster A, Jones C, Chowdhury S I: A bovine    herpesvirus type 1 mutant virus specifying a carboxyl-terminal    truncation of glycoprotein E is defective in anterograde neuronal    transport in rabbits and calves. Journal of virology 2008,    82(15):7432-7442.-   8. Wei H, Wang Y, Chowdhury S I: Bovine herpesvirus type 1 (BHV-1)    UL49.5 luminal domain residues 30 to 32 are critical for MHC-I    down-regulation in virus-infected cells. PloS one 2011,    6(10):e25742.-   9. Brum M C, Coats C, Sangena R B, Doster A, Jones C, Chowdhury S I:    Bovine herpesvirus type 1 (BoHV-1) anterograde neuronal transport    from trigeminal ganglia to nose and eye requires glycoprotein E. J    Neurovirol 2009, 15(2):196-201.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The following statements summarize and describe aspects of theinvention.

Statements:

-   -   1. An expression cassette or expression vector comprising a        nucleic acid segment comprising or consisting essentially of a        sequence with at least 95% sequence identity to SEQ ID NO:2 or        3.    -   2. An expression cassette or expression vector comprising a        nucleic acid segment consisting of a sequence with at least 95%        sequence identity to SEQ ID NO:2 or 3.    -   3. A host cell comprising the expression cassette or expression        vector of statement 1 or 2.    -   4. A method of manufacturing a polypeptide or peptide comprising        culturing a host cell comprising the expression cassette or        expression vector of statement 1 or 2 while the host cell        expresses the polypeptide or peptide from the expression        cassette or expression vector.    -   5. The method of statement 4, wherein the polypeptide or peptide        is an antigen.    -   6. A polypeptide or peptide comprising a sequence with at least        95% sequence identity to any of SEQ ID NO:1, 4-44, or 45.    -   7. A polypeptide or peptide comprising or consisting essentially        of a sequence selected from SEQ ID NO:1, 4-44, or 45.    -   8. The polypeptide or peptide of statement 6 or 7, consisting of        a sequence selected from SEQ ID NO:1, 4-44, or 45.    -   9. The polypeptide or peptide of any of statements 6-8, wherein        the polypeptide or peptide is an antigenic polypeptide or        peptide.    -   10. The polypeptide or peptide of any of statements 6-9,        immobilized to a solid surface.    -   11. The polypeptide or peptide of any of statements 6-10,        covalently attached to a label.    -   12. A composition comprising at least one polypeptide or peptide        antigen comprising sequence with at least 95% sequence identity        to any of SEQ ID NO:1, 4-44, or 45.    -   13. A composition comprising at least one polypeptide or peptide        antigen comprising or consisting essentially of a sequence        selected from SEQ ID NO:1, 4-44, or 45.    -   14. A composition comprising at least one polypeptide or peptide        antigen consisting of a sequence selected from SEQ ID NO:1,        4-44, or 45.    -   15. The composition of any of statements 12-14, further        comprising a carrier.    -   16. The composition of any of statements 12-15, formulated for        administration to an animal.    -   17. An antibody that selectively binds to at least one        polypeptide or peptide antigen comprising a sequence with at        least 95% sequence identity to any of SEQ ID NO:1, 4-44, or 45.    -   18. An antibody that selectively binds to at least one        polypeptide or peptide antigen comprising or consisting        essentially of a sequence selected from SEQ ID NO:1, 4-44, or        45.    -   19. A antibody that selectively binds to an epitope within at        least one polypeptide or peptide antigen comprising or        consisting essentially of a sequence selected from SEQ ID NO:1,        4-44, or 45.    -   20. A antibody that selectively binds to at least one        polypeptide or peptide antigen that comprises an epitope        consisting of a sequence selected from SEQ ID NO:1, 4-44, or 45.    -   21. A device comprising a solid surface and at least one        polypeptide or peptide comprising or consisting essentially of a        sequence with at least 95% sequence identity to any of SEQ ID        NO:1, 4-44, or 45.    -   22. A device comprising a solid surface and at least one        polypeptide or peptide antigen comprising or consisting        essentially of a sequence selected from SEQ ID NO:1, 4-44, or        45.    -   23. A device comprising a solid surface and at least one        polypeptide or peptide antigen consisting of a sequence selected        from SEQ ID NO:1, 4-44, or 45.    -   24. The device of any of statements 21-23, where the solid        surface comprises a chip, strip, paper, microtiter plate, bead,        test tube, slide, or filter.    -   25. The device of any of statements 21-24, where the solid        surface comprises glass, plastic, cellulose, ethylcellulose,        methylcellulose, paper, nitrocellulose, hydroxypropylcellulose,        hydroxypropyl methylcellulose, polystyrene, polyethylene, lipid        polydiacetylene (PDA), polydimethylsiloxane, nylon, rayon,        cotton, teflon, mica, sephadex, sepharose, polyacrylonitrile,        glass-fiber paper, gold, silicon, silica, or combinations        thereof.    -   26. A device comprising a solid surface and at least antibody        that selectively binds to at least one polypeptide or peptide        antigen comprising a sequence with at least 95% sequence        identity to any of SEQ ID NO:1, 4-44, or 45.    -   27. A device comprising a solid surface and at least antibody        that selectively binds to at least one polypeptide or peptide        antigen comprising or consisting essentially of a sequence        selected from SEQ ID NO:1, 4-44, or 45.    -   28. A device comprising a solid surface and at least antibody        that selectively binds to at least one polypeptide or peptide        antigen consisting of a sequence selected from SEQ ID NO:1,        4-44, or 45.    -   29. The device of any of statements 26-28, where the solid        surface comprises a chip, strip, paper, microtiter plate, bead,        test tube, slide, or filter.    -   30. The device of any of statements 26-29, where the solid        surface comprises glass, plastic, cellulose, ethylcellulose,        methylcellulose, paper, nitrocellulose, hydroxypropylcellulose,        hydroxypropyl methylcellulose, polystyrene, polyethylene, lipid        polydiacetylene (PDA), polydimethylsiloxane, nylon, rayon,        cotton, teflon, mica, sephadex, sepharose, polyacrylonitrile,        glass-fiber paper, gold, silicon, silica, or combinations        thereof.    -   31. A method comprising:        -   (a) contacting a test sample with at least one polypeptide            or peptide to form an assay mixture, where the at least one            polypeptide or peptide comprises or consists essentially of            a sequence with at least 95% sequence identity to any of SEQ            ID NO:1, 4-44, or 45; and        -   (b) detecting or measuring whether a complex between the at            least one polypeptide or peptide and antibodies is present            in the assay mixture.    -   32. A method comprising:        -   (a) applying a test sample to a device comprising a solid            surface and at least one polypeptide or peptide to form an            assay mixture, where the at least one polypeptide or peptide            comprises or consists essentially of a sequence with at            least 95% sequence identity to any of SEQ ID NO:1, 4-44, or            45; and        -   (b) detecting or measuring whether a signal from the assay            mixture indicates that antibodies are present in the test            sample.    -   33. A method comprising:        -   (a) applying a test sample to a device comprising a solid            surface and an antibody that selectively binds to at least            one polypeptide or to form an assay mixture, where the at            least one polypeptide or peptide comprises or consists            essentially of a sequence with at least 95% sequence            identity to any of SEQ ID NO:1, 4-44, or 45; and        -   (b) detecting or measuring whether a signal from the assay            mixture indicates that at least one of the polypeptides or            the peptide antigens are present in the test sample.    -   34. The method of any of statements 31-33, wherein the at least        one polypeptide or peptide is a polypeptide antigen or a peptide        antigen.    -   35. The method of any of statement 31-34, wherein the at least        one polypeptide or peptide comprises or consists essentially of        a sequence selected from SEQ ID NO:1, 4-44, or 45.    -   36. The method of any of statement 31-35, the polypeptide or        peptide consists of a sequence selected from SEQ ID NO:1, 4-44,        or 45.    -   37. The method of any of statements 32-36, where the solid        surface comprises a chip, strip, paper, microtiter plate, bead,        test tube, slide, gel, or filter.    -   38. The method of any of statements 32-37, where the solid        surface comprises glass, plastic, cellulose, ethylcellulose,        methylcellulose, paper, nitrocellulose, hydroxypropylcellulose,        hydroxypropyl methylcellulose, polystyrene, polyethylene, lipid        polydiacetylene (PDA), polydimethylsiloxane, polyacrylamide,        nylon, rayon, cotton, teflon, mica, sephadex, sepharose,        polyacrylonitrile, glass-fiber paper, gold, silicon, silica, or        combinations thereof.    -   39. The method of any of statements 31-38, where detecting or        measuring comprises contacting the polypeptide, peptide,        antibody, or a combination thereof with a binding entity.    -   40. The method of statement 39, where the binding entity        comprises at least one label.    -   41. The method of statement 39 or 40, where the binding entity        comprises at least one label that emits a detectable signal.    -   42. The method of any of statements 38-41, where the at least        label is selected from an enzyme, a fluorophore, chromophore,        radioisotope, enzymatic substrate, enzymatic tag, antibody,        chemiluminescent molecule, electroluminescent molecule,        magnetism, electron transmitter, electron dense molecule,        affinity label, or a combination thereof.    -   43. The method of any of statements 31-42, where detecting or        measuring comprises measuring an amount of color, enzymatic        product, radioactivity, chemiluminescence, electroluminescence,        electricity, or a combination thereof.    -   44. The method of any of statements 31-43, where detecting or        measuring comprises measuring optical density of at least a        portion of the device (or the solid surface).    -   45. The method of any of statements 31-44, where detecting or        measuring comprises measuring an optical density or more than        one optical density.    -   46. The method of any of statements 31-45, where detecting or        measuring comprises measuring the optical density of at least        one test sample assay to produce a test sample optical density,        and comparing the test sample optical density to an optical        density of one or more negative control.    -   47. The method of any of statements 31-45, where detecting or        measuring a signal comprises measuring the optical density of at        least one test sample to produce a test sample optical density,        and comparing the test sample optical density to an optical        density of one or more negative control using one or both of the        following algorithms:

${{Percent}\mspace{14mu}{inhibition}} = {\left( \frac{{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} - {{OD}\mspace{14mu}{sample}}}{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right) \times 100}$${S\text{/}N\mspace{14mu}{ratio}} = {\left( \frac{{OD}\mspace{14mu}{sample}}{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right).}$

-   -   48. A kit comprising a instructions for use of the kit        components, and any of the following separately packaged        components:        -   (a) at least one polypeptide or peptide comprising or            consisting essentially of a sequence with at least 95%            sequence identity to any of SEQ ID NO:1, 4-44, or 45;        -   (b) a binding entity that specifically binds to at least one            polypeptide or peptide comprising or consisting essentially            of a sequence with at least 95% sequence identity to any of            SEQ ID NO:1, 4-44, or 45;        -   (c) a secondary binding entity that specifically binds to at            least one polypeptide or peptide comprising or consisting            essentially of a sequence with at least 95% sequence            identity to any of SEQ ID NO:1, 4-44, or 45;        -   (d) a label or a reagent for developing a signal from a            label; or        -   (e) any combination thereof.    -   49. A kit comprising the device of any of statements 21-30, and        instructions for using the device.    -   50. The kit of statement 48, comprising a series of devices.    -   51. The kit of statement 48 or 49, wherein each device is        separately and/or sterilely packaged.

The specific methods, compositions, devices, and kits described hereinare representative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “an antigen” or “anantibody” includes a plurality (for example, a solution of antigens, asolution of antigens, a solution of antibodies or a series of antibodypreparations) of such antigens or antibodies, and so forth. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed:
 1. A method for determining whether an animal isinfected with a Bovine herpesvirus type 1 (BoHV-1), vaccinated with arecombinant BoHV-1 triple mutant virus (BoHV-1 tmv), or uninfected witha Bovine herpesvirus type 1 (BoHV-1) comprising: (a) contacting a testsample from said animal with at least one polypeptide or peptide to forman assay mixture, where the at least one polypeptide or peptide consistsof a sequence selected from the group consisting of SEQ ID NO:1, 4-44,and 45; and (b) detecting or measuring whether a complex between the atleast one polypeptide or peptide and antibodies is present in the assaymixture, and where detecting or measuring comprises contacting thecomplex, or a polypeptide, a peptide, or an antibody in the complex witha binding entity, wherein the binding entity comprises at least onelabel.
 2. The method of claim 1, wherein the at least one polypeptide orpeptide consists of SEQ ID NO:1.
 3. The method of claim 1, wherein theat least one polypeptide or peptide is immobilized on a solid surface.4. The method of claim 3, where the solid surface comprises a chip,strip, paper, microliter plate, bead, test tube, slide, gel, or filter.5. The method of claim 3, where the solid surface comprises glass,plastic, cellulose, ethylcellulose, methylcellulose, paper,nitrocellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose,polystyrene, polyethylene, lipid polydiacetylene (PDA),polydimethylsiloxane, polyacrylamide, nylon, rayon, cotton, teflon,mica, sephadex, sepharose, polyacrylonitrile, glass-fiber paper, gold,silicon, silica, or combinations thereof.
 6. The method of claim 1,where the binding entity comprises at least one label that emits adetectable signal.
 7. The method of claim 1, where the at least label isselected from an enzyme, fluorophore, chromophore, radioisotope,enzymatic substrate, enzymatic tag, antibody, chemiluminescent molecule,electroluminescent molecule, magnetism, electron transmitter, electrondense molecule, affinity label, or a combination thereof.
 8. The methodof claim 1, where detecting or measuring comprises measuring an amountof color, enzymatic product, radioactivity, chemiluminescence,electroluminescence, electricity, or a combination thereof.
 9. Themethod of claim 1, where detecting or measuring comprises measuringoptical density.
 10. The method of claim 1, where detecting or measuringcomprises measuring optical density of the assay mixture, or a signalproduced by a label present in the assay mixture.
 11. The method ofclaim 1, where detecting or measuring comprises measuring the opticaldensity of at least one assay mixture to produce an assay mixtureoptical density, and comparing the assay mixture optical density to anoptical density of one or more negative control, one or more positivecontrol, or both.
 12. The method of claim 1, where detecting ormeasuring comprises measuring the optical density of at least one assaymixture to produce an assay mixture optical density, and comparing theassay mixture optical density to an optical density of one or morenegative control using one or both of the following algorithms:${{Percent}\mspace{14mu}{inhibition}} = {\left( \frac{{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} - {{OD}\mspace{14mu}{sample}}}{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right) \times 100}$${S\text{/}N\mspace{14mu}{ratio}} = \left( \frac{{OD}\mspace{14mu}{sample}}{{OD}\mspace{14mu}{negative}\mspace{14mu}{control}} \right)$wherein: OD negative control is the optical density of the negativecontrol; and OD sample is the optical density of the assay mixtureoptical density.
 13. An expression cassette or expression vectorcomprising a nucleic acid segment encoding a polypeptide or peptidewhich consists of a sequence selected from the group consisting of SEQID NO:1, 4-44, and 45 and further comprises a heterologous promoter. 14.An expression cassette or expression vector comprising a nucleic acidsegment comprising a sequence with at least 95% sequence identity to SEQID NO:2 or 3 and further comprises a heterologous promoter.
 15. A devicefor determining whether an animal is infected with a Bovine herpesvirustype 1 (BoHV-1), vaccinated with a recombinant BoHV-1 triple mutantvirus (BoHV-1 tmv), or uninfected with a Bovine herpesvirus type 1(BoHV-1) comprising a solid surface and at least one polypeptide orpeptide consists of a sequence selected from the group consisting of SEQID NO:1, 4-44, and
 45. 16. A kit comprising the device of claim 15, andinstructions for using the device.
 17. The device of claim 15, whereinthe at least one polypeptide or peptide consists of SEQ ID NO:1.
 18. Akit for determining whether an animal is infected with a Bovineherpesvirus type 1 (BoHV-1), vaccinated with a recombinant BoHV-1 triplemutant virus (BoHV-1 tmv), or uninfected with a Bovine herpesvirus type1 (BoHV-1) comprising a instructions for use of the kit components, andany of the following separately packaged components: (a) at least onepolypeptide or peptide consists of a sequence selected from the groupconsisting of SEQ ID NO:1, 4-44, and 45; (b) a binding entity thatspecifically binds to at least one polypeptide or peptide consists of asequence selected from the group consisting of SEQ ID NO:1, 4-44, and45; (c) a secondary binding entity that specifically binds to at leastone polypeptide or peptide consists of a sequence selected from thegroup consisting of SEQ ID NO:1, 4-44, and 45; (d) a label or a reagentfor developing a signal from a label; or (e) any combination thereof.19. The kit of claim 18, wherein the at least one polypeptide or peptideconsists of SEQ ID NO:1.